CA2080482C - Isolated promoter and terminator of elongation factor ef-1.alpha. - Google Patents
Isolated promoter and terminator of elongation factor ef-1.alpha. Download PDFInfo
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Abstract
The promoter region of the A. gossypii gene which encodes translation elongation factor EF-1.alpha. is described.
The promoter can be employed for protein synthesis.
The promoter can be employed for protein synthesis.
Description
ISOLATED PROMOTER AND TERMINATOR OF ELONGATION FACTOR EF-la Description The present invention relates to a promoter region from Ashbya gossypii (= A. gossypii), to fungi which have been genetically modified with this promoter region, and to the use thereof.
A. gossypii is employed for the production of vitamin BZ by fermentation. It is desirable to extend the use of the fermentation technology available for A. gossypii by producing protein products using genetic engineering methods. An expression system for A. gossypii is required for this purpose. Systems of this type have already been described for some higher ascomycetes such as Aspergillus niger (Rambosek and Leach, CRC Critical Reviews in Biotechnology 6 (1987), 357-393). By contrast, to date no experience in the area of genetic engineering is available with the hemiascomycete A. gossypii, which is the sole representative of its genus.
An essential component of a system for the expression of genes which code for a required product is the so-called promoter region which is composed of 1) a functional promoter which is indispensable for transcription of the gene, and 2) the 5' non-coding region (between promoter and translation start) which is necessary for transla-tion after transcription into mRNA.
The invention relates to the promoter region of the A. gossypii TEF gene which encodes translation elongation factor EF-:l« (= TEF-la).
This gene is very strongly expressed and there-fore has a very efficient promoter region.
The promoter region obtained according to the invention has the nucleotide sequence indicated in sequence listing No. 1. Since the limits of the functional regions of a novel and sequenced promoter region, which have the ability to initiate transcription and translation, can be defined well at the 3' end and less well at the 5' end, it cannot be ruled out that the natural promoter region of A. gossypii gene differs slightly in length from the indicated sequence.
The invention further relates to the terminator region of the A. gossypii TEF gene which encodes trans-lation elongation factor EF-la (= TEF-la).
The terminator region can be used for efficient termination of transcription.
The terminator region obtained according to the invention has the nucleotide sequence indicated in sequence listing No. 2, position 1513-2095. 3'-Terminal truncations of this sequence are also suitable as trans-cription terminator.
The terminator region can be used in conjunction with the TEF-promoter region or with other homologous or heterologous promoters.
The invention. further relates to fungi which contain the above mentioned promoter region or parts thereof and/or the above mentioned terminator region or parts thereof.
The promoter region can be inserted, in particu-lar, into the following fungi:
Ashbya gossypii,, species closely related to Ashbya, such as, in particular, Eremothecium ashbyi and genera unre-lated to Ashbya, such as, in particular, Aspergillus and Neurospora.
The novel promoter region can be prepared a) by cloning the gene for translation elongation factor EF-la (TEF gene) from A. gossypii including adjoining DNA sequences and subsequently cleaving, b) by fusion of A. gossypii DNA fragments to an open reading frame of a promoterless gene which is selectable in A. gossypii, isolation of strongly expressing transformants and subsequent selection of the TEF promoter, 2~~~:~8~
w - 3 - O.Z. 0050/41686 c) by chemical synthesis using known methods.
The novel promoter region makes it possible, together with suitable vector systems, to bring about overexpression of homologous and heterologous proteins in A. gossypii and other fungi. This may entail, for ex-ample, constitutively enhanced expression of genes of vitamin BZ biosynthesis, and of genes which are respons-ible for overproduction of vitamin B2, or the overexpres-sion and isolation of proteins which are of economic importance. It is furthermore possible with the aid of the novel promoter region to utilize the post-transcript-ional modification potential (e.g. glycosilation) of A. gossypii, which in some circumstances differs from that of other fungi. Since it is not possible to prepare all heterologous proteins in sufficient amounts by, the-systems hitherto used, such as Aspergillus or ' Saccharomyces, the development of expression systems with the efficient TEF promoter region for novel host organ-isms (for example A. gossypii in this case) is of great importance.
EXAMPLES
1. Isolation of the Ashbya gossypii TEF gene DNA isolated from A. gossypii mycelium was cut with the restriction endonucleases EcoRI and BamHI.
DNA fragments which harbor the TEF gene or parts thereof were identified after separation of the restriction fragments according to size in an agarose gel electrophoresis and subsequent hybrid-ization with a 32P-labeled heterologous TEF gene probe. The TEF gene probe comprises nucleotides 363 to 1235 of the 1377 bp-long open reading frame of the S. cerevisiae TEF2 gene (Schirmaier and Philippsen, EMBO J. 3 (1984), 3311-3315). A
4.6 kb-long EcoRI fragment and a 6.4 kb-long BamHI
fragment hybridized with the heterologous TEF gene probe. Fragments with lengths in these ranges were eluted from from agarose gels, cloned into the 2n~~~~?
- 4 - O.Z. 0050/41686 vector pUCB (Vieira and Messing, Gene 19 (1982), 259-268) which had been cut with EcoRI or HamHI, and transformed into E. coli. The clones with TEF DNA
were identified by colony hybridization (Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 72 (1975), 3961-3965) using the 32P-labeled heterologous probe.
The positive clones contained either the 4.6 kb-long EcoRI fragment or the 6.4 kb-long BamHI fragment.
The two clones overlap in a 2.1 kb region which carries the homology with the TEF gene probe and which was sequenced (sequence No. 2). This 2.1 kb-long fragment contains the open reading frame of 1377 bp, 136 by of the 5'-non-coding region and 582 by of the 3'-non-coding region. Beyond the EcoRI cleavage site, a further 278 by of the_ 5'-non-coding region were determined up to a HindIII~
cleavage site. Subsequently, the promoter region was isolated as 403 bp-long HindIII/HincII fragment which, besides the 379 by in front of the start codon, also harbors the first 24 by of the open reading frame of the TEF gene, and was employed for the constructions of pAG-100 and pAG-101 (sequence No. 1).
2. Plasmid constructions a) The vector pAG-1 (Fig. 1) (deposited DSM 6010), a derivative.of the vector pEX4, was prepared as described by Ernst and Chan, J. Bacteriol. 163 - (1985), 8-14. pAG-1 contains a 1.7 kb Sall fragment with the kanamycin-resistance gene, which codes for the aminoglycoside phosphotrans ferase (APH(3')I), of the transposon Tn903. In the original pEX4 construct, initially the 1695 by PvuII fragment of Tn903 (Oka et al., J. Mol. Biol. 147 (1981), 217-226) was ligated into a plasmid with filled-in Sall cleavage sites. The SalI cleavage sites wars: retained in this way, and the resistance gene can be isolated ~(!~~~J!~~~:
- 5 - O.Z. 0050/41686 as 1.7 kb SalI fragment. pAG-1 contains the Saccharomyces cerevisiae ARS elements ARS1 and 2~ ARS and undergoes autonomous replication in Ashbya gossypii.
b) pAG-2 (Fig. 2). The 1.7 kb SalI fragment with the kanamycin-resistance gene was cut out of pAG-1 and inserted into the SalI cleavage site of the S. cerevisiae E. coli shuttle vector XEp24 (Botstein et al., Gene 8 (1979), 17-24; New England Biolabs Inc., Beverly, MA, USA, 1988-1989 Catalog, 112-113). The structure of the newly produced plasmid -- pAG-2 - was checked by re-striction endonuclease mapping, using the XhoI
cleavage site which is located in the 1.7 kb SalI_ fragment to check the orientation of the insert.-pAG-2 contains the Saccharomyces cerevisiae ARS ' element 2Es ARS and undergoes autonomous replica-tion in Ashbya gossypii.
c) pAG-100 (Fig. 3). A 403 bp-long HindIII/HincII
fragment which contains the promoter region and the first 24 by of the open reading frame of the gene for translation elongation factor EF-la (TEF
gene) from A. gossypii was inserted, after the protruding ends had been filled in, into the XhoI
cleavage site of pAG-2 which is located 30 by in the 3' direction behind the translation start of the kanamycin-resistance gene. The orientation of the fragment in the plasmid pAG-100 produced in this way was checked by restriction endonuclease mapping with HindIII. Insertion of the 403 be-long fragment resulted in replacement of the 10 N-terminal amino acids of APH(3')I by the first 8 amino acids of A. gossypii translation elongation factor EF-la. Deletion or replacement of the first 19 amino acids of APH(3')I by other amino acids does not result in loss of activity (Chen and Fukuhara, Gene 69 (1988), 181-192). The (.: _.. 'v ;.,' - 6 - O.Z. 0050/41686 sequence of the SalI fragment after insertion of the TEF promoter region is shown in sequence No. 2. pAG-100 contains the Saccharomyces cerevisiae ARS element 2~ ARS and undergoes autonomous replication in Ashbya gossypii.
d) pAG-5 (Fig. 4). The 1.7 kb fragment with the kanamycin-resistance gene from pAG-1 was sub-cloned into the SalI cleavage site of pBR322 (Bolivar et al., Gene 2 (1977), 95-113). The resulting plasmid - p,TL3A - contains in the pBR322 portion one BamHI cleavage site and one EcoRI cleavage site so that pJL3A is decomposed by double digestion into a 375 by and a 5688 by fragment. The large fragment was ligated to a_ 2.1 kb EcoRI/HamHI A. gossypii fragment which-contains the open reading frame of the gene for _' translation elongation factor EF-1a (TEF gene) (sequence No. 1). The resulting plasmid was called pAG-5. pAG-5 contains no Saccharomyces cerevisiae ARS elements.
e) pAG-101 (Fig. 5). The 403 by HindIII/HincII
fragment with the promoter region and the first 24 by of the open reading frame of the TEF gene from A. gossypii was inserted into the Xhol cleavage site located in the open reading frame of the kanamycin-resistance gene, as described for the construction of pAG-100. The plasmid - produced in this way was called pAG-101. pAG-101 contains no Saccharomyces cerevisiae ARS
elements.
f) pBRS1871 (precursor plasmid for TEF promoter-lacZ
fusion): A 3113 bp-long Pstl fragment from the plasmid pMC1871 (Shapira et al., Gene, 25 (1983), 71-82) was cloned into the PstI cleavage site of the plasmid pBRS+ (Short et al., Nucleic Acid Res . , 16 ( 1988 ) , 7583-7600 ) . The fragment harbors ~~~.~~ ~w - 7 - O.Z. 0050/41686 the open reading frame of the lacZ gene from E. coli (Ralnins et al., EMBO J., 2 (1983), 593-597) which lacks the first seven codons.
g) pPLl (Fig. 6). pBRS1871 was linearized at the SmaI cleavage site in front of the lacZ gene. A
1500 by HincII fragment which harbors the TEF
promoter and adjoining sequences including the first eight codons of the TEF gene from A. gossypii was cloned into the linearized plasmid. This resulted in an open reading frame which codes for a ,B-galactosidase whose first seven amino acids are replaced by the first eight amino acids of the EF-la from A. gossypii. It was possible to isolate TEF promoter-lacZ fusions with regions of various lengths of the TEF~_ promoter from this plasmid.
h) pPL2 (Fig. 9). pBRS1871 was linearized at the SmaI cleavage site in front of the lacZ gene. A
294 bp-long Rsal/HincII fragment which contains parts of the TEF promoter (270 bp) and the first eight codons of the TEF gene from A. gossypii (24 bp) was cloned into the linearized plasmid.
i) pPL3 (Fig. 10). pBRS1871 was linearized at the SmaI cleavage site in front of the lacZ gene. A
239 bp-long HaeIII/HincTI fragment which contains the first eight codons of the TEF gene (24 bp) and 215 by of the regions, located in the 5' direction in front of the start codon, of the non-translated region was cloned into the linear-ized plasmid.
j) pPL4 (Fig. 11). pHRS1871 was linearized at the SmaI cleavage site in front of the lacZ gene. A
158 bp-long EcoRI/HincII fragment which contains the first eight codons of the TEF gene and 134 by of the regions, located in the 5' direction in front of the start codon, of the non-translated region was cloned into the linearized plasmid.
~~~~3~~~
- 8 - O.Z. 0050/41686 k) pAG-110 (Fig. 7). Cleavage of pPLl with XbaI and SalI resulted in isolation of a 4600 by fragment which harbors the fusion of the 1500 bp-long TEF
promoter fragment with the lacZ gene. After the protruding ends had been filled in, this fragment was cloned into the filled-in BamHI cleavage site of pAG-100.
1) pAG-111 (Fig. 8). Cleavage of pPLl with HindIII
resulted in isolation of a 3509 bp-long fragment.
The TEF promoter region is truncated by 1100 by in this fragment. It thus corresponds to the promoter region which in pAG-100, pAG-101, pAG-110 and pAG-111 controls transcription of the 6418 resistance gene. After the protruding ends.
had been filled in, the 3509 bp-long fragment was cloned into the filled-in BamHI cleavage site of ' pAG-100.
m) pAG-112 (Fig. 12). After cleavage of pPL2 with XbaI and SalI, a 3392 bp-long fragment which harbors the fusion of the 294 bp-long promoter fragment with the lacZ gene was isolated and, after the protruding ends had been filled in, was inserted into the filled-in BamHI cleavage site of the plasmid pAG-100.
n) pAG-113 (Fig. 13). After cleavage of pPL3 with XbaI and Sall, a 3337 bp-long fragment which harbors the fusion of the 239 bp-long promoter fragment with the lacZ gene was isolated and, after the protruding ends had been filled in, was inserted into the filled-in BamHI cleavage site of the plasinid pAG-100.
o) pAG-114 (Fig. 14). After cleavage of pPL4 with HindIII, a 3273 bp-long fragment which harbors the fusion of the 158 bp-long promoter fragment with the lacZ gene was isolated and, after the protruding ends had been filled in, was inserted into the filled-in BamHI cleavage site of the n - 9 - O.Z. 0050/41686 plasmid pAG-100.
p) pAG-115 (Fig. 15). After cleavage of pBRS 1871 with BamHI, a 3069 bp-long fragment which harbors the open reading frame of the lacZ gene with the first seven codons of the open reading frame being missing and no promoter fragment being fused in front of the open reading frame was isolated. This fragment was inserted into the BamHI cleavage site of the plasmid pAG-100.
g) pAG-120.pBIIRS (Short et al., Nucleic Acid Res.
16 (1988), 7583-7600) was cleaved with SspI and ScaI, and a 2084 bp-long fragment was isolated.
YEP24 (Botstein et al., Gene 8 (1979), 1?-24) was cleaved with Scal and ClaI, and a fragment Z5 2782 by in size was isolated. This was ligated,- .
after the protruding ends had been filled in, to ., the 2084 bp-long ScaI/SspI fragment from pBIIRS-so that a complete ampicillin-resistance gene was produced again (in pBIIRS- arid YEP24, ScaI cuts in the ampicillin-resistance gene).
r) pAG-121.pAG-100 was cut with SalI and HindIII, and a 669 bp-long fragment which harbors part of the 6418-resistance gene was isolated. This was cloned into the SAlI/HindIII cut plasmid pBIISR+
(Short et al., Nucleic Acid Res. 16 (1988), 7583-7600).
s) pAG-122.pAG-100 was cut with HindIII, and a - 940 bp-long fragment which harbors part of the 6418-resistance gene under the control of the TES
promoter. This was inserted into the HindIII-cut plasmid pAG-121 in such a way that a complete 6418-resistance gene was produced. Transformation of this plasmid into E. coli permits transformant selection on kanamycin-containing medium.
t) pAG-123.pAG-122 was cut with SalI~and BamHI, and a 1639 bp-long fragment which harbors the G418-resistance gene under the control. of the TEF
4' -~ 10 - O.Z. 0050/41686 promoter was isolated. This was inserted into the ScaI-cut plasmid pAG-120, which made selection of E. coli transformants on kanamycin-containing medium possible.
u) pAG-130.pBIIRS+ (Short et al., Nucleic Acid.
Res. 16 (1988), 7583-7600) was cleaved with HindIII and HincII, and the 403 bp-long HindIII/HincII TEF promoter fragment was inserted.
v) pAG-131. An HaeIII/AccI fragment which is 260 by in size and which contains 25 nucleotides of the 3' end of the TEF gene and regions adjacent thereto in the 3' direction (terminator fragment) was isolated from the clone which harbors the fragment 2.1 kb in size, which contains the TEF-' gene, of genomic A. gossypii DNA. After the protruding ends had been filled in, this fragment was inserted into the plasmid pBIIRS- which had been cleaved with HincII (Short et al., Nucleic Acid Res. 16 (1988), 7583-7600).
w) pAG-132. pag-130 was cut with ScaI and XhoI, and a fragment 2248 by in size was isolated. pAG-131 was likewise cleaved with Scal and XhoI, and a fragment 1442 by in size was isolated and was ligated to the 2248 by fragment from pAG-103 in such a way that a complete ampicillin-resistance gene was produced anew.
x) M13PT. pAG-132 was cleaved with BamHI, and a fragment which is 752 by in size and which contains the fusion of TEF promoter fragment and TEF terminator fragment was isolated. This was cloned into the BamHI cleavage site of M13mp9.
y) M13PT1, M13PT2, M13PT3.
M13PT was modified by oligonucleotide-directed mutagenesis (Rramer et al., Nucl. acid. Res. 24 (1984), 9441-9556) so as to produce an Scal cleavage site behind the stop codon of the TEF
~ :~ ~~ ~J ~. Vie' ;
w - 11 - O.Z. 0050/41686 gene (in the terminator fragment) and an NcoI
cleavage site (M13PT1), an NsiI cleavage site (M13PT2) or an SphI cleavage site (M13PT3) in the start codon of the TEF gene (in the promoter fragment) (Fig. 17).
z) pAG-201. pAG-202, pAG-203 (Fig. 18).
M13PT1, M13PT2 and M13PT3 were cleaved with BamHI, and the fragment which is 751 by in size and has promoter region and terminator region of the TEF gene was isolated from the cleavage. This TEF signal sequence was inserted into the BamHI
cleavage site of the plasmid pAG-123 to yield the plasmid pAG-201. The same method was used to construct the plasmid pAG-202 from M13PT2 and the plasmid pAG-203 from M13PT3.
3. Transformation of A. gossypii with TEF promoter. ' region plasmids The transformations were carried out in accordance with the following scheme:
- Inoculate 200 ml of MA2 with about 1-2x10' spores - Incubate in flasks with baffles at 27°C and 350 rpm for 32-40 h.
- Remove mycelium by filtration with suction and wash lx in 30 ml of H20 - Determine fresh weight (about 2-3 g) - Suspend mycelium in 30 ml of SD and incubate at 30°C in a shaker for 30 min.
- Suspend mycelium in 5-10 ml of SPEZ per g fresh weight - Incubate'in a water bath shaker at 30°C, check protoplast formation under the microscope (a degree of protoplast formation of more than 90 should be reached after 30 min.) - Filter) protoplast suspension through glass filter (Schott, porosity 1) - Centrifuge filtrate for 5 min. (Sorvall SM24 rotor, 1800 rpm) - 12 - O.Z. 0050/41686 - Wash sediment lx in 20 ml of ST and lx in 20 ml of STC
- Suspend protoplasts in 20 ml of STC and determine titer in a counter - After centrifugation, resuspend protoplasts to a density of 4x108/ml in STC
- Add 100 u1 of protoplast suspension to DNA in a maximum of 15 ~1 of TE and mix (amounts of DNA:
for replicating TEF promoter region plasmids: 1-10 gig; for integrative transformation with linearized TEF promoter region plasmids: 15-gig) - Incubate at room temperature for 15 min.
Cautiously add 1 ml of PTC40 and mix by inversion_ 15 - Centrifuge for 5 min. (Heraeus Biofuge A,-1500 rpm) - Cautiously remove supernatant, and suspend sediment in 1 ml of SMTCI
- Incubate at 27°C for 3 h., mix about every 20 45 min. by inversion - After centrifugation, suspend sediments in 1 ml of SM
- Mix suspension with 9 ml of SMA2 top layer and place on SMA2 plate (20 ml of SMA2 agar per plate) - Incubate plates at 27°C for 18 h.
- Place 6418 layer on plates (0.54 ml of 6418 stock solution + 0.46 ml of H20 + 6 ml of 0.5 ~ of agarose (in HZO, preheated to 42°C)) - Incubate plates further at 27°C, transformants are visible after 2-3 days in the case of repli-cating plasmids, and after 3-6 days in the case of integration Media and solutions Media: MA2: Peptone (Gibco casein hydrolyzate (No. 140) . 10 g/1 Yeast extract (Gibco) . 1 g/1 - 13 - O.Z. 0050/41686 Glucose . 10 g/1 myo-Inositol . 0.3 g/1 SMA2-agar: Sorbitol . 1 M
Peptone . 10 g/1 Yeast extract . 1 g/1 Glucose . 20 g/1 myo-Inositol . 0.3 g/1 Agar (Gibco) . 12 g/1 SMA2 top layer: As SMA2 agar, 0.8 % agarose in place of agar Solutions: SD: 1M sorbitol; 50 mM dithiothreitol SPEZ: 1M sorbitol; 10 mM Na phosphate buffer pH 5.8;
10 mM EDTA; 2 mg/ml Zymolyase 20 T
(Seikagaku Kogyo Co., Tokyo) r .
ST: 1M sorbitol; 10 mM tris-C1 pH 8 STC: 1M sorbitol; 10 mM tris-Cl pH 8;
10 mM CaCl2 TE: 10 mM tris-C1; 1 mM EDTA
PTC40: 40 % (w/v.) polyethylene glycol 4000 (Merck); 10 mM tris-C1 pH 8; 10 mM
CaCl2 SMTCI: 50 % SM (see below); 50 % STC;
0.03 g/1 myo-inositol SM: SO % 2 M sorbitol; 50 $ MA2 6418 stock solution: 2 0 m g l m 1 G 4 1 8 (Geneticin, Gibco) in Ha0 4. Results of transformation with TEF promoter region plasmids The results of various transformations carried out as in Example 3 ase compiled in Table 1. In all the experiments, tranaformants were selected with a 6418 concentration of 0.3 mg/ml per transformation plate. Growth of A. gossypii mycelium is completely inhibited at this 6418 concentration. On transforma-tion with the recombinant DNA, vectors pAG-1 and 2~~~~~~~;
- 14 - O.Z. 0050/41686 pAG-2, in which the 6418-resistance gene is under the control of the original bacterial promoter and not under the control of the TEF promoter region, no transformants are produced at this concentration. In order to obtain transformants with these recombinant DNA vectors, the 6418 concentration must not exceed 0.1 mg/ml per transformation plate. At this concen-tration up to 80 ~ of the colonies which appear are not transformants.
TABLE 1: Transformation results Experi- Plasmid DNA per Transform- Transformants meet transf., ants per per viable ~g ~g of DNA protoplasts 1 pAG-1 10 0 0 1 pAG-2 10 0 0 -1 pAG-100 10 10 1.2 x 10-°
2 pAG-100 0.1 10 1.6 x 10-5 3 pAG-10 0 1 3 3 . 4 x 10-' 3 pAG-101, 20 0.05 1.I x 10-5 linear ized with BamHI
, 5. Results of transformation with lacZ plasmids In order to investigate the functioning ability of the TEF promoter further, derivatives of the plasmid pAG-100 in which the gene for p-galactosid-ase from E. coli (lacZ gene) is under the control of the TEF promoter were constructed. For this, various regions of the promoter region of the TEF gene were fused in front of the open reading frame of the lacZ
gene, with the first seven codons of the lacZ gene being replaced by the first eight codons of the TEF
gene. The plasmid pAG-110 harbors an approximately 1.5 kb-long HincII TEF promoter fragment in front of the lacZ gene and the plasmid pAG-111 the 403 bp-long HindIII/HincII TEF promoter fragment which has already been employed for the constructions of pAG-100 and pAG-101. The plasmid pAG-112 harbors a ~~~G~~'a%
- 15 - O.Z. 0050/41686 294 bp-long TEF promoter fragment, plasmid pAG-113 a 239 bp-long TEF promoter fragment and pAG-114 a 158 bp-long TEF promoter fragment.
In addition, pAG-115 which harbors the open reading frame of the lacZ gene without fusion to a promoter fragment was constructed as control plasmid.
After transformation of these plasmids into A. gossypii, the expression of the lacZ gene was checked using a color test. The ~-galactosidase encoded by the lacZ gene cleaves X-Gal (5-bromo-4-chloro-3-indoyl p-D-galactoside) to the blue dye 5-bromo-4-chloroindigo.
pAG-110, pAG-111 and pAG-112 transformants formed blue colonies on medium which contains X-Gal (Miller, Experiments in Molecular Genetics, Cold Spring Harbor, New York 1972, 48) in a concentration of 100 ~g/ml. No-blue coloration was visible in the case of transformants. ' which contained pAG-113, pAG-114 or pAG-115.
Fig. 16 shows a summary of the various TEF
promoter fragments which were fused in front of the lacZ
gene. A + represents a blue coloration of the colonies on X-Gal-containing medium, a - represents no visible blue coloration.
For a further investigation of ~-galactosidase expression, the p-galactosidase activity of liquid cultures of pAG-110, pAG-111, pAG-112, pAG-113, PAG-114 and pAG-115 transformants was determined. The mycelium was disrupted with glass beads for this (Rose, M.;
Casadaban, M.J. and Botstein, D., Proc. Natl. Acad. Sci.
USA Vol. 78, No. 4 (1981), 2460-2464). 0.5 g of mycelium which had grown in MA2 liquid medium containing 200 ~g/ml G 418 was taken up in 0.1 mM Tris, pH 8.0/20 $ (vol/vol) glycerol/1 mM DTT/1 mM PMSF and, after addition of 0.5 g of glass beads~(diameter 0.45-0.5 mm), frozen away at -20°C. To disrupt the mycelium it was shaken vigorously (Vortex) at 4°C for l5,sec. 12 times. It was subsequently centrifuged at 10000 rpm (Sorvall cooled centrifuge) twice for 20 min. The supernatants were diluted 1:10 and 20~~~°~
- 16 - O.Z. 0050/41686 1:20, respectively, in Z buffer (0.06 M Na2HP0~/0.04 M
NaH2P0~/0.01 M KC1/0.001 M MgS04/0.05 M ~-mercaptoeth-anol). The ~-galactosidase activity in the diluted protein crude extracts was determined by cleavage of o-nitrophenyl p-D-galactopyranoside (Miller, Experiments in Molecular Genetics, Cold Spring Harbor, New York 1972, 353 ff). The enzyme.activity was related to the protein concentration in the crude extract, which was determined by the Bradford method (Bradford, M.M., Anal. Biochem. 72 (1976), 248-254). The results of the ~-galactosidase activity determination are shown in Table 2. The amount of o-nitrophenol (measured as OD4zo) liberated per minute and mg of total protein is indicated.
TABLE 2: ~-Galactosidase expression Plasmid Measurement p-Galactosidase activity No. (relative units, ODazo%mg min.) pAG-110 1 3.62 2 3.54 3 2.45 pAG-111 1 3.07 2 3.29 3 3.63 4 3.16 pAG-112 1 1.89 2 1.90 3 1.79 4 1.75 pAG-113 1 0 pAG-114 1 0 pAG-115 1 0 Sequence listing No. 1:
Sequence type: nucleotide Sequence length: 409 base pairs Strandedness: single strand r r~ ~;
- 17 - O.Z. 0050/41686 Topology: linear Molecule type: genomic DNA
Source: A. gossypii Properties: promoter region AAGCAAAAAT TACGGCTCCT CGCTGCAGAC CTGCGAGCAG GGAAACGCTC CCCTCAGCAG 2=~0 CACTGAGGTT CTTCTTTCAT ATACTTCCTT TTAAAATCTT GCTAGGATAC AGTTCTCACA.360 No. 2 Sequence of the 2.1 kb EcoRI/BamHI fragment with the open reading frame of the TEF gene 5' OdATfQ'CL~ G1CC,CrL~Ct~ ~GlC ,~'aTld~U, CGe'fbCGlf! TGCC~C1~C
crTerTCrrr c~rxrac~rc c.~rT~ errcQacca T~cac~rrcrc ~c~carc 130 140 1sa 1so 1, o lao C9sACdIAEA GSLA~PGt39 TlAOQddAdQ dtI~C~Tfl Al.'GTTG1CL-I vdl~iTTC:dI:
190 200 210 220 ?30 240 GTCG1CTCTC ~7ldCI'C~1C TICCdCeOCt C1C:~TQ AC~Gi'f1'G6 I~GT~i'.GaC
250 260 270 280 2~0 300 AIGbGJI~CCI TCGAGidGIT CC~C~d6CbC G~'OCCG1C! TGCCrAdf,GC Ti'C~'t2Cl~C
- 310 320 330 340 3:4 360 Tack rrrn~au u°rcaacas c~cac~cac~ c~ccr~TC~.e caTacacaTr xc~~rTa~ acrrarcac Ta~ucrx caccrc~crc Tc~TTCacx aracxcc~c uo 4va 4so 460 4~0 4ao ac~caa~a rcaa~cat r~tavcca acTrar~c cnac~vc casrrrcax 4~o soo slo 520 spa s4o ~rroabcrc ~czoccrca crr~cocr ~ra~rcr~ bccaCOCTCn s~cam~c sso sso s7o sao s9o soo caooc:~TCZ rcocrriac ct~ccci~c uoca"crsw x~rrcccar caacaar~Tc 6I0 620 630 6t0 6=0 660 Gl,CPGCC~CCd CTCf~Gi3~C GGCa6d~~ lCI~GGa6lC CeCCLiCrlC
IT~Cl~GC TCCC~C7d CCCTIaG~S 6~TGGCIT3eC TTCC11TCYC C~CfG~C
730 740 7so 7so 770 7ao - 18 - O.Z. 0050/41686 No. 2 (Continuation) 'w'1'CrlCdalC~1 TC~ll1'G~CGC C7~CC1CC~1C GCt~dTGC! ~1GCGCIG GwIG~GWC
190 E00 d14 d20 830 880 ACC.uGCCTG GTGCCCTC~ GGGTaIC~bCC 1RG1'T~GG CClIC11TC~CCCd 850 860 8?0 8E0 890 900 CCrCTCaGaC CuCTC71C3a CGGTTG16~ TfGCGT~'OC IGa GGTxTTGGEl CGQTCG6T CGGC71G1G!'C G1GJICCGCI'C TCbTCJ~GCC aCGT~TGQ'1' 970 9a0 990 1000 1010 1020 GiTIC~TTCC CCCL"11~C TAT G? CC~fC~IGIT GCa"CCaCCaG
1030 lOdO 1050 1060 1070 1080 ~11TIGCiCC aGO~'C1~C 1GC1~'dClbC G~OG°f~ Cll CT;~:CGTC
1090 1100 1110 11~ 1130 lld0 ~IGGlG~ C11161GG111 CCPTlOC6CT dC~llGi LC4CLL'~CC 6~lCOQ'~?
u:~o ueo u~g uav ux~ uuu GaCI~ITCI 1C'CCT1~Q' CaTTQ°f~l'C M~CCC~C G1d181~C1C 2GC~G1?1C
TCTCGGTC! TC~Ci'OC~C b!i'OC11GTD 1GT1'CCdCGa CT!'cT~GiC
1~0 ugp u9p 1300 1310 1320 J1IGMCGbCI GadG~iGEdCiT6 CiaG~C ClIllC1'TCC1' u,IAO~~°t C~dC4C'~Cl '~rGTCBdCT! 'fG~°OC~l~C 1M~CCi~T6? CTCinJGGC 'rPi~0.G~C
1390 1400 IdlO 140 1130 1410 ray r~r~c,~ct ccC~c~ G~cas~c ~eca~cc 1450 . 1160 1170 1110 Id90 1300 ~ICnTC TTGTCAiGTC CCaC81o0CT G~?~CCTC1 CCiBGOCCOC cc~6oQ
151A L'u0 1330 3510 1550 1580 GCt~t lGa6TB~CPC liC3ri1'J~il~ Gdn'C~TGY! Zl°ClICUCa 'TCTC~Ti1'Cd 1570 1u0 1594 1600 1610 1620 bTl~'! ?ITlin?u '1~'t~?d'li't i~3'G1~! CllaA0~1'Gi ?llI~R~S
1630 1610 1650 1564 16'0 1680 T'1TT~CTCG 1GITClTCTC CCCaGaTCCC ~GtTIIIGTG C~C~6T A1T~TGC
crcSaBTCet, T3t3AiTOC! uaxo~?a2d C.roetcTCO~1 xrCQix~,C:a eC90G9CGr 1750 1760 17?0 1780 1190 1800 CQGTC1~C'd8 a:I'G1'C~Aa2 TT(~CC1GCCT ClI~TGCCTC Ca6G~txlGldl T11'GCZCGaC
~Q'GT~ C AaGGITIiCC CaTa1'GC~CP ATCGGCGG<1G allICGPTGC
CACJIGOC~'rCT 1'CC'11~L~ GdCCTO~~CC ?TCCaCTGCl' IGITCICItiG T1CGCGr.TIG
'lTaaCTTTC C1GOCCI~'! ~TOOCGCa ITIsIJSOC1T aTP60Gt'i~ GCtaCGeCl2 1990 2000 2014 ~0~0 2034 1040 CTGIOOCaIGa CGlCCl ~GGaCD00N' iTi~Tl2~ GGDC1CGG4 2050 2060 2090 x030 2090 ~~1TI1'11W GCCaCCOaIC CaTGl'L'GCa? :C~Za? TG11CG~'dG GbTCC 3' 2(~~~~~
- 19 - O.Z. 0050/41686 No. 3 Sequence of the kanamxcin-resistance gene - TEF promoter region fusion (TEF promoter region sequences underlined) 20 30 ;0 50 50 5' CTCGaC'i'C2A ATC~JC C~idCCGAd CAtTCATGG PClaMAA2C c~ITCLTu 7o ao 90 loa 1.o zzo A4CCC1'fXbC AGCOCGC1GG Gf~d(~fGa ATACCCCP~! M2GICClaC ACdCTC~TGA
130 140 150 160 1'0 180 TG(xuCCTC 1GL121t~,bC TGICCfCI'OC Q'CGTGUGa AGC;"CTTCQ GBCPCbTACV
190 200 210 220 2;0 2a0 iGGCCfCUT CCCCC:ATG 'rCGGCGGa AaGTGaGG~ GCCICfCITC A.TCaGaGCIR
250 260 270 280 2~0 300 T~'."GP1GC! GG~ICCaGfTC fI'G31'i'Z'1'G~ A .. TGCCICG~i Ct~I'CTGCt?
'1'c~TCCwaaC l:GCCTGATC TGaT~I~Ca ~.TCac~ rICTTCGaITT ~Caa1 GtCdCCfTGT Gu;TCUUT f~7~Cfi AC~TfGGCa ACaTAaaAIT ATaTGTCIT
470 440 450 660 4i0 4E0 GLiCUTUI 1TACafluC~ G~dTICaaC GGG'1'QnT6 aOCCaTaTfC
AJiCs~G~llaC GTQTGQ'CC
:30 560 570 580 590 600 a 610 620 630 640 6:0 660 570 6a0 690 700 710 720 730 740 750 760 770 7a0 79o aoo slo ago a3a :4o aso aso a7o aao ago 900 GGCCCC6aT! laaTTOCaaC ~fCG~fiOCTC lTf1'lmTCO GTayuIIGC
970 980 990 1000 1010 10x0 i9 1TCTCG~C1 ATCdlCdaT~TC C1TTCT~C Cu~3 1030 1014 1050 1060 1070 10x0 cc~caracr xrrl~crcaa as cc~cccr~c craaTCxxr racacarAc Axcr~ca~ ~aucacocr caa~caara aZC~crrc c~am~a c~TZr~tc II50 1150 2170 ZZaO 1190 1200 CC?lt~'CCTG aT~'GC1TC GdT,CTC1CC ICfOOGi2CC OC~6aauC IGCaTTfC3IC
1210 1210 1230 1240 1250 12b0 CrllTaGiaG aaTaTOG'TCa lTnOr.~Gaa laTaTT4'11°0 a'1'GlQCtTCLTG
C~61TOC 1!'1'CGiI'rOC TGrIIlGTld! ~ aCl~ilrC Ca'IiTi°CG~
1330 1340 1350 136!1 I37A 13a0 Q'C~OC OOCUl~OC ui~'11C OCfiT~T~6 A~CWQra i~ITCfI~C
1390 1400 1~10 1~120 1430 1140 carp ccxoc~ra xaacucrc rcrsucua T~arucca ~c~rx s ua~aC gas rrcrcaax aruscxrar ~c 15!o lsao 133a is4o ipso ipso a tai xo ~~~c~ta r~cxcac~ ariCCaaar 1570 15E0 1590 1600 1610 lbZO
C'!t'GCClfCC TITC~ICl~C OC~TGIG lt~~T~'! CaTTIGGlI AC~T'iR°!
~~~'~~o - 20 - O.Z. 0050/41686 No. 3 (Continuation) 1630 16x0 1650 1660 16'0 1580 Cl~2bTC G:ITTGaTLI TC~ITI~TTC ldTIA~TTOC dG3T:~'.dT:T G~TGCTCGa2 GbGr~1TTC!' ~IITCaGadT? CCIT7IdTTOC TTGg3.lUQ' (~UGaGGI2 "3CCCTGACT
1750 1760 1770 1780 1,'90 1800 'fG~CGCG~CG GxGQ'tTGT TGa1T1,1TC GLICPrffCC I'G.1 .G':~d~lG C1TGG1TG
CCGTCTPCC C'~dCMCGCB C,~CCGTTC~ TG~GC~ dk~CTi~L ATCbCC.IdCT
GOTCC~CT1 GUC~GQ CTGTC~CC GTCOCTCCCT cad C~T&
G OIGCt~TTCa G~tGTl2 C~CTUt~ GC~PrZTTC ~CCBt~CdCA c~TUOCGC:T
aTrp'G~!' T~GTCaCG 1TC8Tt1~1CC CCTATrGGC Cl'GlCCCTGC
GCGCT~C1 G~ifir~iT C~TCc~ TI,C~CIWT G~~ITCflCCO TTGC~CC
CGGl!lLGG TxlC 3~
Xeys to Figures 1 to 8 Fig. 1: Plasmid pAG-1. ARS: S. cerevisiae ARS1 sequence;
2 micron; EcoRI fragment of the S, cerevisiae 2~
plasmid with replication origin; URA3:
S. cerevisiae URA3 gene; G418r: 6418 (kanamycin) resistance; black arrow: S. cerevisiae cycl-13 promoter; black box: S. cerivisiae CYC1 termin-ator; white arrows represent the direction of transcription.
Fig. 2: Plasmid pAG-2. amp: ampicillin resistance;
2 micron: EcoRI fragment of the S. cerevisiae 2~
plasmids with replication origin; URA3:
S. cerevisiae URA3 gene; G418r: 6418 (kanamycin) resistance; ORI: arigin of plasmid replication in E. coli; white arrows represent the direction of transcription.
Fig. 3: Plasmid pAG-100. amp: ampicillin resistance;
2 micron: EcoRI fragment of the S. cerevisiae 2~
plasmid with replication origin; URA3:
S. cerevisiae URA3 gene; G418r: 6418 (kanamycin) resistance; ORI: origin of plasmid replication in 2~c~.~'~r~~~
- 21 - O.Z. 0050/41686 E. coli; black arrow: A. gossypii DNA fragment with TEF promoter region; white arrows represent the direction of transcription.
Fig. 4: Plasmid pAG-5. amp: ampicillin resistance; G418r:
(kanamycin) resistance; ORI: origin of plasmid replication in E. coli; TEF: A. gossypii EcoRI/
BamHI fragment with ORF for the translation elongation factor; white arrows represent the direction of transcription.
Fig. 5: Plasmid pAG-101. amp: ampicillin resistance;
G418r: 6418 (kanamycin) resistance; ORI: origin of plasmid replication in E. coli; TEF:
A. gossypii EcoRI/BamHI fragment with ORF for the translation elongation factor; black arrow:
A. gossypii DNA fragment with TEF promoter-region; white arrows represent the direction of ' transcription.
Fig. 6: Plasmid pPLl. amp: ampicillin-resistance gene;
M13+: replication origin for single-stranded DNA
isolation; ori: origin for plasmid replication in E. coli; lacZ: E. coli lacZ gene; prom: 1500 by A. gossypii DNA fragment with the TEF promoter region.
Fig. 7: Plasmid pAG-110. 2u: EcoRI fragment of the S. cerevisiae 2~ plasmid with replication origin;
URA3: S. cerevisiae URA3 gene; prom: 1500 by A. gossypii DNA fragment with the TEF promoter region; lacZ: E. coli laxZ gene; G418r: 6418 (kanamycin) resistance gene; ari: origin for plasmid replication in E. coli; amp: ampicillin-resistance gene; black arrow: 1500 by A. gossypii DNA fragment with the TEF promoter region; white arrows represent the direction of transcription.
Fig. 8: Plasmid pAG-110. 2p: EcoRI fragment of the S. cerevisiae 2~ plasmid with replication origin;
URA3: S. cerevisiae URA3 gene; prom: 403 by A. gossypii DNA fragment with the TEF promoter 2~~~~~~
- 22 - O.Z. 0050/41686 region; lacZ: E. coli lacZ gene; G418r: 6418 (kanamycin) resistance gene; ori: origin for plasmid replication in E. coli; amp: ampicillin-resistance gene; black arrow: A. gossypii DNA
fragment with the TEF promoter region; white arrows represent the direction of transcription.
Fig. 9: Plasmid pPL2. amp: ampicillin-resistance gene;
M13+: replication origin foz single-stranded DNA
isolation; ori: origin for plasmid replication in E. coli; lacZ; E. coli lacZ gene; prom:
294 by A. gossypii DNA fragment with a part of the TEF promoter region (2?0 bp).
Fig. 10: Plasmid pPL3. amp: ampicillin-resistance gene;
M13+: replication origin for single-stranded DNA
isolation; ori: origin for plasmid replication--:
in E. coli; lacZ; E, coli lacZ gene; prom:.
239 by A. gossypii DNA fragment with a part of the TEF promoter region (215 bp).
Fig. 11: Plasmid pPL4. amp: ampicillin-resistance gene;
M13+: replication origin for single-stranded DNA
isolation; ori: origin for plasmid replication in E. coli; lacZ; E. coli lacZ gene; prom:
158 by A. gossypii DNA fragment with a part of the TEF promoter region (134 bp).
Fig. 12: Plasmid pAG-112. 2~: EcoRI fragment of the S. cerevisiae 2~ plasmid with replication origin; URA3: S. cerevisiae URA3 gene; prom:
294 by A. gossypii DNA fragment with the TEF
promoter region; lacZ: E. coli lacZ gene; G418r:
6418 (kanamycin) resistance gene; ori: origin for plasmid replication in E. coli; amp: ampi-cillin-resistance gene; black arrow: A. gossypii DNA fragment with the TEF promoter region; white arrows represent the direction of transcription.
Fig. 13: Plasmid pAG-113. 2~: EcoRI fragment of the S. cerevisiae 2;a plasmid with replication origin; URA3: S. c~revisiae URA3 gene; prom:
- 23 - O.Z. 0050/41686 239 by A. gossypii DNA fragment with the TEF
promoter region; lacZ: E. coli lacZ gene; G418r:
6418 (kanamycin) resistance gene; ori: origin for plasmid replication in E. coli; amp: ampi-cillin-resistance gene; black arrow: A. gossypii DNA fragment With the TEF promoter region; white arrows represent the direction of transcription.
Fig. 14: Plasmid pAG-114. 2~: EcoRI fragment of the S. cerevisiae 2~. plasmid with replication origin; URA3: S. cerevisiae URA3 gene; prom:
158 by A. gossypii DNA fragment with the TEF
promoter region; lacZ: E. coli lacZ gene; G418r:
6418 (kanamycin) resistance gene; ori: origin for plasmid replication in E. coli; amp: ampi-cillin-resistance gene; black arrow: A. gossypii-DNA fragment with the TEF promoter region; white ' arrows represent the direction of transcription.
FIg. 15: Plasmid pAG-115. 2~s: EcoRI fragment of the S. cerevisiae 2~ plasmid with replication origin; URA3: S. cerevisiae URA3 gene; lacZ:
E. coli lacZ gene; G418r: 6418 (kanamycin) resistance gene; ori: origin for plasmid repli cation .in E. coli; amp: ampicillin-resistance gene; white arrows represent the direction of transcription.
Fig. 16: TEF promoter fragments of the ,9-galactosidase expression plasmids.
Fig. I7: Nucleotide sequence in the ATG region and in the terminator region of MI3PT1, M13PT2, M13PT3, pAG-201, pAG-202, pAG-203.
Fig. 18: Plasmid pAG-201, pAG-202, pAG-203. 2~: ECORI
fragment of the S. cerevisiae 2~ plasmid with replication origin: prom, term: 751 by A. gossypii DNA fragment with the TEF promoter-terminator fusian. G418r:G418 (kanamycin) resistance gene; ori: origin point of plasmid replication in E. coli; amp:
~~, u~ ~; ~~ 'p. ~~ f.i - 24 - O.Z. 0050/41686 ampicillin-resistance gene; white arrows repre-sent the direction of transcription.
Fig. 19: Nucleotide sequence of the fusion of the promoter and terminator of the TEF gene.
A. gossypii is employed for the production of vitamin BZ by fermentation. It is desirable to extend the use of the fermentation technology available for A. gossypii by producing protein products using genetic engineering methods. An expression system for A. gossypii is required for this purpose. Systems of this type have already been described for some higher ascomycetes such as Aspergillus niger (Rambosek and Leach, CRC Critical Reviews in Biotechnology 6 (1987), 357-393). By contrast, to date no experience in the area of genetic engineering is available with the hemiascomycete A. gossypii, which is the sole representative of its genus.
An essential component of a system for the expression of genes which code for a required product is the so-called promoter region which is composed of 1) a functional promoter which is indispensable for transcription of the gene, and 2) the 5' non-coding region (between promoter and translation start) which is necessary for transla-tion after transcription into mRNA.
The invention relates to the promoter region of the A. gossypii TEF gene which encodes translation elongation factor EF-:l« (= TEF-la).
This gene is very strongly expressed and there-fore has a very efficient promoter region.
The promoter region obtained according to the invention has the nucleotide sequence indicated in sequence listing No. 1. Since the limits of the functional regions of a novel and sequenced promoter region, which have the ability to initiate transcription and translation, can be defined well at the 3' end and less well at the 5' end, it cannot be ruled out that the natural promoter region of A. gossypii gene differs slightly in length from the indicated sequence.
The invention further relates to the terminator region of the A. gossypii TEF gene which encodes trans-lation elongation factor EF-la (= TEF-la).
The terminator region can be used for efficient termination of transcription.
The terminator region obtained according to the invention has the nucleotide sequence indicated in sequence listing No. 2, position 1513-2095. 3'-Terminal truncations of this sequence are also suitable as trans-cription terminator.
The terminator region can be used in conjunction with the TEF-promoter region or with other homologous or heterologous promoters.
The invention. further relates to fungi which contain the above mentioned promoter region or parts thereof and/or the above mentioned terminator region or parts thereof.
The promoter region can be inserted, in particu-lar, into the following fungi:
Ashbya gossypii,, species closely related to Ashbya, such as, in particular, Eremothecium ashbyi and genera unre-lated to Ashbya, such as, in particular, Aspergillus and Neurospora.
The novel promoter region can be prepared a) by cloning the gene for translation elongation factor EF-la (TEF gene) from A. gossypii including adjoining DNA sequences and subsequently cleaving, b) by fusion of A. gossypii DNA fragments to an open reading frame of a promoterless gene which is selectable in A. gossypii, isolation of strongly expressing transformants and subsequent selection of the TEF promoter, 2~~~:~8~
w - 3 - O.Z. 0050/41686 c) by chemical synthesis using known methods.
The novel promoter region makes it possible, together with suitable vector systems, to bring about overexpression of homologous and heterologous proteins in A. gossypii and other fungi. This may entail, for ex-ample, constitutively enhanced expression of genes of vitamin BZ biosynthesis, and of genes which are respons-ible for overproduction of vitamin B2, or the overexpres-sion and isolation of proteins which are of economic importance. It is furthermore possible with the aid of the novel promoter region to utilize the post-transcript-ional modification potential (e.g. glycosilation) of A. gossypii, which in some circumstances differs from that of other fungi. Since it is not possible to prepare all heterologous proteins in sufficient amounts by, the-systems hitherto used, such as Aspergillus or ' Saccharomyces, the development of expression systems with the efficient TEF promoter region for novel host organ-isms (for example A. gossypii in this case) is of great importance.
EXAMPLES
1. Isolation of the Ashbya gossypii TEF gene DNA isolated from A. gossypii mycelium was cut with the restriction endonucleases EcoRI and BamHI.
DNA fragments which harbor the TEF gene or parts thereof were identified after separation of the restriction fragments according to size in an agarose gel electrophoresis and subsequent hybrid-ization with a 32P-labeled heterologous TEF gene probe. The TEF gene probe comprises nucleotides 363 to 1235 of the 1377 bp-long open reading frame of the S. cerevisiae TEF2 gene (Schirmaier and Philippsen, EMBO J. 3 (1984), 3311-3315). A
4.6 kb-long EcoRI fragment and a 6.4 kb-long BamHI
fragment hybridized with the heterologous TEF gene probe. Fragments with lengths in these ranges were eluted from from agarose gels, cloned into the 2n~~~~?
- 4 - O.Z. 0050/41686 vector pUCB (Vieira and Messing, Gene 19 (1982), 259-268) which had been cut with EcoRI or HamHI, and transformed into E. coli. The clones with TEF DNA
were identified by colony hybridization (Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 72 (1975), 3961-3965) using the 32P-labeled heterologous probe.
The positive clones contained either the 4.6 kb-long EcoRI fragment or the 6.4 kb-long BamHI fragment.
The two clones overlap in a 2.1 kb region which carries the homology with the TEF gene probe and which was sequenced (sequence No. 2). This 2.1 kb-long fragment contains the open reading frame of 1377 bp, 136 by of the 5'-non-coding region and 582 by of the 3'-non-coding region. Beyond the EcoRI cleavage site, a further 278 by of the_ 5'-non-coding region were determined up to a HindIII~
cleavage site. Subsequently, the promoter region was isolated as 403 bp-long HindIII/HincII fragment which, besides the 379 by in front of the start codon, also harbors the first 24 by of the open reading frame of the TEF gene, and was employed for the constructions of pAG-100 and pAG-101 (sequence No. 1).
2. Plasmid constructions a) The vector pAG-1 (Fig. 1) (deposited DSM 6010), a derivative.of the vector pEX4, was prepared as described by Ernst and Chan, J. Bacteriol. 163 - (1985), 8-14. pAG-1 contains a 1.7 kb Sall fragment with the kanamycin-resistance gene, which codes for the aminoglycoside phosphotrans ferase (APH(3')I), of the transposon Tn903. In the original pEX4 construct, initially the 1695 by PvuII fragment of Tn903 (Oka et al., J. Mol. Biol. 147 (1981), 217-226) was ligated into a plasmid with filled-in Sall cleavage sites. The SalI cleavage sites wars: retained in this way, and the resistance gene can be isolated ~(!~~~J!~~~:
- 5 - O.Z. 0050/41686 as 1.7 kb SalI fragment. pAG-1 contains the Saccharomyces cerevisiae ARS elements ARS1 and 2~ ARS and undergoes autonomous replication in Ashbya gossypii.
b) pAG-2 (Fig. 2). The 1.7 kb SalI fragment with the kanamycin-resistance gene was cut out of pAG-1 and inserted into the SalI cleavage site of the S. cerevisiae E. coli shuttle vector XEp24 (Botstein et al., Gene 8 (1979), 17-24; New England Biolabs Inc., Beverly, MA, USA, 1988-1989 Catalog, 112-113). The structure of the newly produced plasmid -- pAG-2 - was checked by re-striction endonuclease mapping, using the XhoI
cleavage site which is located in the 1.7 kb SalI_ fragment to check the orientation of the insert.-pAG-2 contains the Saccharomyces cerevisiae ARS ' element 2Es ARS and undergoes autonomous replica-tion in Ashbya gossypii.
c) pAG-100 (Fig. 3). A 403 bp-long HindIII/HincII
fragment which contains the promoter region and the first 24 by of the open reading frame of the gene for translation elongation factor EF-la (TEF
gene) from A. gossypii was inserted, after the protruding ends had been filled in, into the XhoI
cleavage site of pAG-2 which is located 30 by in the 3' direction behind the translation start of the kanamycin-resistance gene. The orientation of the fragment in the plasmid pAG-100 produced in this way was checked by restriction endonuclease mapping with HindIII. Insertion of the 403 be-long fragment resulted in replacement of the 10 N-terminal amino acids of APH(3')I by the first 8 amino acids of A. gossypii translation elongation factor EF-la. Deletion or replacement of the first 19 amino acids of APH(3')I by other amino acids does not result in loss of activity (Chen and Fukuhara, Gene 69 (1988), 181-192). The (.: _.. 'v ;.,' - 6 - O.Z. 0050/41686 sequence of the SalI fragment after insertion of the TEF promoter region is shown in sequence No. 2. pAG-100 contains the Saccharomyces cerevisiae ARS element 2~ ARS and undergoes autonomous replication in Ashbya gossypii.
d) pAG-5 (Fig. 4). The 1.7 kb fragment with the kanamycin-resistance gene from pAG-1 was sub-cloned into the SalI cleavage site of pBR322 (Bolivar et al., Gene 2 (1977), 95-113). The resulting plasmid - p,TL3A - contains in the pBR322 portion one BamHI cleavage site and one EcoRI cleavage site so that pJL3A is decomposed by double digestion into a 375 by and a 5688 by fragment. The large fragment was ligated to a_ 2.1 kb EcoRI/HamHI A. gossypii fragment which-contains the open reading frame of the gene for _' translation elongation factor EF-1a (TEF gene) (sequence No. 1). The resulting plasmid was called pAG-5. pAG-5 contains no Saccharomyces cerevisiae ARS elements.
e) pAG-101 (Fig. 5). The 403 by HindIII/HincII
fragment with the promoter region and the first 24 by of the open reading frame of the TEF gene from A. gossypii was inserted into the Xhol cleavage site located in the open reading frame of the kanamycin-resistance gene, as described for the construction of pAG-100. The plasmid - produced in this way was called pAG-101. pAG-101 contains no Saccharomyces cerevisiae ARS
elements.
f) pBRS1871 (precursor plasmid for TEF promoter-lacZ
fusion): A 3113 bp-long Pstl fragment from the plasmid pMC1871 (Shapira et al., Gene, 25 (1983), 71-82) was cloned into the PstI cleavage site of the plasmid pBRS+ (Short et al., Nucleic Acid Res . , 16 ( 1988 ) , 7583-7600 ) . The fragment harbors ~~~.~~ ~w - 7 - O.Z. 0050/41686 the open reading frame of the lacZ gene from E. coli (Ralnins et al., EMBO J., 2 (1983), 593-597) which lacks the first seven codons.
g) pPLl (Fig. 6). pBRS1871 was linearized at the SmaI cleavage site in front of the lacZ gene. A
1500 by HincII fragment which harbors the TEF
promoter and adjoining sequences including the first eight codons of the TEF gene from A. gossypii was cloned into the linearized plasmid. This resulted in an open reading frame which codes for a ,B-galactosidase whose first seven amino acids are replaced by the first eight amino acids of the EF-la from A. gossypii. It was possible to isolate TEF promoter-lacZ fusions with regions of various lengths of the TEF~_ promoter from this plasmid.
h) pPL2 (Fig. 9). pBRS1871 was linearized at the SmaI cleavage site in front of the lacZ gene. A
294 bp-long Rsal/HincII fragment which contains parts of the TEF promoter (270 bp) and the first eight codons of the TEF gene from A. gossypii (24 bp) was cloned into the linearized plasmid.
i) pPL3 (Fig. 10). pBRS1871 was linearized at the SmaI cleavage site in front of the lacZ gene. A
239 bp-long HaeIII/HincTI fragment which contains the first eight codons of the TEF gene (24 bp) and 215 by of the regions, located in the 5' direction in front of the start codon, of the non-translated region was cloned into the linear-ized plasmid.
j) pPL4 (Fig. 11). pHRS1871 was linearized at the SmaI cleavage site in front of the lacZ gene. A
158 bp-long EcoRI/HincII fragment which contains the first eight codons of the TEF gene and 134 by of the regions, located in the 5' direction in front of the start codon, of the non-translated region was cloned into the linearized plasmid.
~~~~3~~~
- 8 - O.Z. 0050/41686 k) pAG-110 (Fig. 7). Cleavage of pPLl with XbaI and SalI resulted in isolation of a 4600 by fragment which harbors the fusion of the 1500 bp-long TEF
promoter fragment with the lacZ gene. After the protruding ends had been filled in, this fragment was cloned into the filled-in BamHI cleavage site of pAG-100.
1) pAG-111 (Fig. 8). Cleavage of pPLl with HindIII
resulted in isolation of a 3509 bp-long fragment.
The TEF promoter region is truncated by 1100 by in this fragment. It thus corresponds to the promoter region which in pAG-100, pAG-101, pAG-110 and pAG-111 controls transcription of the 6418 resistance gene. After the protruding ends.
had been filled in, the 3509 bp-long fragment was cloned into the filled-in BamHI cleavage site of ' pAG-100.
m) pAG-112 (Fig. 12). After cleavage of pPL2 with XbaI and SalI, a 3392 bp-long fragment which harbors the fusion of the 294 bp-long promoter fragment with the lacZ gene was isolated and, after the protruding ends had been filled in, was inserted into the filled-in BamHI cleavage site of the plasmid pAG-100.
n) pAG-113 (Fig. 13). After cleavage of pPL3 with XbaI and Sall, a 3337 bp-long fragment which harbors the fusion of the 239 bp-long promoter fragment with the lacZ gene was isolated and, after the protruding ends had been filled in, was inserted into the filled-in BamHI cleavage site of the plasinid pAG-100.
o) pAG-114 (Fig. 14). After cleavage of pPL4 with HindIII, a 3273 bp-long fragment which harbors the fusion of the 158 bp-long promoter fragment with the lacZ gene was isolated and, after the protruding ends had been filled in, was inserted into the filled-in BamHI cleavage site of the n - 9 - O.Z. 0050/41686 plasmid pAG-100.
p) pAG-115 (Fig. 15). After cleavage of pBRS 1871 with BamHI, a 3069 bp-long fragment which harbors the open reading frame of the lacZ gene with the first seven codons of the open reading frame being missing and no promoter fragment being fused in front of the open reading frame was isolated. This fragment was inserted into the BamHI cleavage site of the plasmid pAG-100.
g) pAG-120.pBIIRS (Short et al., Nucleic Acid Res.
16 (1988), 7583-7600) was cleaved with SspI and ScaI, and a 2084 bp-long fragment was isolated.
YEP24 (Botstein et al., Gene 8 (1979), 1?-24) was cleaved with Scal and ClaI, and a fragment Z5 2782 by in size was isolated. This was ligated,- .
after the protruding ends had been filled in, to ., the 2084 bp-long ScaI/SspI fragment from pBIIRS-so that a complete ampicillin-resistance gene was produced again (in pBIIRS- arid YEP24, ScaI cuts in the ampicillin-resistance gene).
r) pAG-121.pAG-100 was cut with SalI and HindIII, and a 669 bp-long fragment which harbors part of the 6418-resistance gene was isolated. This was cloned into the SAlI/HindIII cut plasmid pBIISR+
(Short et al., Nucleic Acid Res. 16 (1988), 7583-7600).
s) pAG-122.pAG-100 was cut with HindIII, and a - 940 bp-long fragment which harbors part of the 6418-resistance gene under the control of the TES
promoter. This was inserted into the HindIII-cut plasmid pAG-121 in such a way that a complete 6418-resistance gene was produced. Transformation of this plasmid into E. coli permits transformant selection on kanamycin-containing medium.
t) pAG-123.pAG-122 was cut with SalI~and BamHI, and a 1639 bp-long fragment which harbors the G418-resistance gene under the control. of the TEF
4' -~ 10 - O.Z. 0050/41686 promoter was isolated. This was inserted into the ScaI-cut plasmid pAG-120, which made selection of E. coli transformants on kanamycin-containing medium possible.
u) pAG-130.pBIIRS+ (Short et al., Nucleic Acid.
Res. 16 (1988), 7583-7600) was cleaved with HindIII and HincII, and the 403 bp-long HindIII/HincII TEF promoter fragment was inserted.
v) pAG-131. An HaeIII/AccI fragment which is 260 by in size and which contains 25 nucleotides of the 3' end of the TEF gene and regions adjacent thereto in the 3' direction (terminator fragment) was isolated from the clone which harbors the fragment 2.1 kb in size, which contains the TEF-' gene, of genomic A. gossypii DNA. After the protruding ends had been filled in, this fragment was inserted into the plasmid pBIIRS- which had been cleaved with HincII (Short et al., Nucleic Acid Res. 16 (1988), 7583-7600).
w) pAG-132. pag-130 was cut with ScaI and XhoI, and a fragment 2248 by in size was isolated. pAG-131 was likewise cleaved with Scal and XhoI, and a fragment 1442 by in size was isolated and was ligated to the 2248 by fragment from pAG-103 in such a way that a complete ampicillin-resistance gene was produced anew.
x) M13PT. pAG-132 was cleaved with BamHI, and a fragment which is 752 by in size and which contains the fusion of TEF promoter fragment and TEF terminator fragment was isolated. This was cloned into the BamHI cleavage site of M13mp9.
y) M13PT1, M13PT2, M13PT3.
M13PT was modified by oligonucleotide-directed mutagenesis (Rramer et al., Nucl. acid. Res. 24 (1984), 9441-9556) so as to produce an Scal cleavage site behind the stop codon of the TEF
~ :~ ~~ ~J ~. Vie' ;
w - 11 - O.Z. 0050/41686 gene (in the terminator fragment) and an NcoI
cleavage site (M13PT1), an NsiI cleavage site (M13PT2) or an SphI cleavage site (M13PT3) in the start codon of the TEF gene (in the promoter fragment) (Fig. 17).
z) pAG-201. pAG-202, pAG-203 (Fig. 18).
M13PT1, M13PT2 and M13PT3 were cleaved with BamHI, and the fragment which is 751 by in size and has promoter region and terminator region of the TEF gene was isolated from the cleavage. This TEF signal sequence was inserted into the BamHI
cleavage site of the plasmid pAG-123 to yield the plasmid pAG-201. The same method was used to construct the plasmid pAG-202 from M13PT2 and the plasmid pAG-203 from M13PT3.
3. Transformation of A. gossypii with TEF promoter. ' region plasmids The transformations were carried out in accordance with the following scheme:
- Inoculate 200 ml of MA2 with about 1-2x10' spores - Incubate in flasks with baffles at 27°C and 350 rpm for 32-40 h.
- Remove mycelium by filtration with suction and wash lx in 30 ml of H20 - Determine fresh weight (about 2-3 g) - Suspend mycelium in 30 ml of SD and incubate at 30°C in a shaker for 30 min.
- Suspend mycelium in 5-10 ml of SPEZ per g fresh weight - Incubate'in a water bath shaker at 30°C, check protoplast formation under the microscope (a degree of protoplast formation of more than 90 should be reached after 30 min.) - Filter) protoplast suspension through glass filter (Schott, porosity 1) - Centrifuge filtrate for 5 min. (Sorvall SM24 rotor, 1800 rpm) - 12 - O.Z. 0050/41686 - Wash sediment lx in 20 ml of ST and lx in 20 ml of STC
- Suspend protoplasts in 20 ml of STC and determine titer in a counter - After centrifugation, resuspend protoplasts to a density of 4x108/ml in STC
- Add 100 u1 of protoplast suspension to DNA in a maximum of 15 ~1 of TE and mix (amounts of DNA:
for replicating TEF promoter region plasmids: 1-10 gig; for integrative transformation with linearized TEF promoter region plasmids: 15-gig) - Incubate at room temperature for 15 min.
Cautiously add 1 ml of PTC40 and mix by inversion_ 15 - Centrifuge for 5 min. (Heraeus Biofuge A,-1500 rpm) - Cautiously remove supernatant, and suspend sediment in 1 ml of SMTCI
- Incubate at 27°C for 3 h., mix about every 20 45 min. by inversion - After centrifugation, suspend sediments in 1 ml of SM
- Mix suspension with 9 ml of SMA2 top layer and place on SMA2 plate (20 ml of SMA2 agar per plate) - Incubate plates at 27°C for 18 h.
- Place 6418 layer on plates (0.54 ml of 6418 stock solution + 0.46 ml of H20 + 6 ml of 0.5 ~ of agarose (in HZO, preheated to 42°C)) - Incubate plates further at 27°C, transformants are visible after 2-3 days in the case of repli-cating plasmids, and after 3-6 days in the case of integration Media and solutions Media: MA2: Peptone (Gibco casein hydrolyzate (No. 140) . 10 g/1 Yeast extract (Gibco) . 1 g/1 - 13 - O.Z. 0050/41686 Glucose . 10 g/1 myo-Inositol . 0.3 g/1 SMA2-agar: Sorbitol . 1 M
Peptone . 10 g/1 Yeast extract . 1 g/1 Glucose . 20 g/1 myo-Inositol . 0.3 g/1 Agar (Gibco) . 12 g/1 SMA2 top layer: As SMA2 agar, 0.8 % agarose in place of agar Solutions: SD: 1M sorbitol; 50 mM dithiothreitol SPEZ: 1M sorbitol; 10 mM Na phosphate buffer pH 5.8;
10 mM EDTA; 2 mg/ml Zymolyase 20 T
(Seikagaku Kogyo Co., Tokyo) r .
ST: 1M sorbitol; 10 mM tris-C1 pH 8 STC: 1M sorbitol; 10 mM tris-Cl pH 8;
10 mM CaCl2 TE: 10 mM tris-C1; 1 mM EDTA
PTC40: 40 % (w/v.) polyethylene glycol 4000 (Merck); 10 mM tris-C1 pH 8; 10 mM
CaCl2 SMTCI: 50 % SM (see below); 50 % STC;
0.03 g/1 myo-inositol SM: SO % 2 M sorbitol; 50 $ MA2 6418 stock solution: 2 0 m g l m 1 G 4 1 8 (Geneticin, Gibco) in Ha0 4. Results of transformation with TEF promoter region plasmids The results of various transformations carried out as in Example 3 ase compiled in Table 1. In all the experiments, tranaformants were selected with a 6418 concentration of 0.3 mg/ml per transformation plate. Growth of A. gossypii mycelium is completely inhibited at this 6418 concentration. On transforma-tion with the recombinant DNA, vectors pAG-1 and 2~~~~~~~;
- 14 - O.Z. 0050/41686 pAG-2, in which the 6418-resistance gene is under the control of the original bacterial promoter and not under the control of the TEF promoter region, no transformants are produced at this concentration. In order to obtain transformants with these recombinant DNA vectors, the 6418 concentration must not exceed 0.1 mg/ml per transformation plate. At this concen-tration up to 80 ~ of the colonies which appear are not transformants.
TABLE 1: Transformation results Experi- Plasmid DNA per Transform- Transformants meet transf., ants per per viable ~g ~g of DNA protoplasts 1 pAG-1 10 0 0 1 pAG-2 10 0 0 -1 pAG-100 10 10 1.2 x 10-°
2 pAG-100 0.1 10 1.6 x 10-5 3 pAG-10 0 1 3 3 . 4 x 10-' 3 pAG-101, 20 0.05 1.I x 10-5 linear ized with BamHI
, 5. Results of transformation with lacZ plasmids In order to investigate the functioning ability of the TEF promoter further, derivatives of the plasmid pAG-100 in which the gene for p-galactosid-ase from E. coli (lacZ gene) is under the control of the TEF promoter were constructed. For this, various regions of the promoter region of the TEF gene were fused in front of the open reading frame of the lacZ
gene, with the first seven codons of the lacZ gene being replaced by the first eight codons of the TEF
gene. The plasmid pAG-110 harbors an approximately 1.5 kb-long HincII TEF promoter fragment in front of the lacZ gene and the plasmid pAG-111 the 403 bp-long HindIII/HincII TEF promoter fragment which has already been employed for the constructions of pAG-100 and pAG-101. The plasmid pAG-112 harbors a ~~~G~~'a%
- 15 - O.Z. 0050/41686 294 bp-long TEF promoter fragment, plasmid pAG-113 a 239 bp-long TEF promoter fragment and pAG-114 a 158 bp-long TEF promoter fragment.
In addition, pAG-115 which harbors the open reading frame of the lacZ gene without fusion to a promoter fragment was constructed as control plasmid.
After transformation of these plasmids into A. gossypii, the expression of the lacZ gene was checked using a color test. The ~-galactosidase encoded by the lacZ gene cleaves X-Gal (5-bromo-4-chloro-3-indoyl p-D-galactoside) to the blue dye 5-bromo-4-chloroindigo.
pAG-110, pAG-111 and pAG-112 transformants formed blue colonies on medium which contains X-Gal (Miller, Experiments in Molecular Genetics, Cold Spring Harbor, New York 1972, 48) in a concentration of 100 ~g/ml. No-blue coloration was visible in the case of transformants. ' which contained pAG-113, pAG-114 or pAG-115.
Fig. 16 shows a summary of the various TEF
promoter fragments which were fused in front of the lacZ
gene. A + represents a blue coloration of the colonies on X-Gal-containing medium, a - represents no visible blue coloration.
For a further investigation of ~-galactosidase expression, the p-galactosidase activity of liquid cultures of pAG-110, pAG-111, pAG-112, pAG-113, PAG-114 and pAG-115 transformants was determined. The mycelium was disrupted with glass beads for this (Rose, M.;
Casadaban, M.J. and Botstein, D., Proc. Natl. Acad. Sci.
USA Vol. 78, No. 4 (1981), 2460-2464). 0.5 g of mycelium which had grown in MA2 liquid medium containing 200 ~g/ml G 418 was taken up in 0.1 mM Tris, pH 8.0/20 $ (vol/vol) glycerol/1 mM DTT/1 mM PMSF and, after addition of 0.5 g of glass beads~(diameter 0.45-0.5 mm), frozen away at -20°C. To disrupt the mycelium it was shaken vigorously (Vortex) at 4°C for l5,sec. 12 times. It was subsequently centrifuged at 10000 rpm (Sorvall cooled centrifuge) twice for 20 min. The supernatants were diluted 1:10 and 20~~~°~
- 16 - O.Z. 0050/41686 1:20, respectively, in Z buffer (0.06 M Na2HP0~/0.04 M
NaH2P0~/0.01 M KC1/0.001 M MgS04/0.05 M ~-mercaptoeth-anol). The ~-galactosidase activity in the diluted protein crude extracts was determined by cleavage of o-nitrophenyl p-D-galactopyranoside (Miller, Experiments in Molecular Genetics, Cold Spring Harbor, New York 1972, 353 ff). The enzyme.activity was related to the protein concentration in the crude extract, which was determined by the Bradford method (Bradford, M.M., Anal. Biochem. 72 (1976), 248-254). The results of the ~-galactosidase activity determination are shown in Table 2. The amount of o-nitrophenol (measured as OD4zo) liberated per minute and mg of total protein is indicated.
TABLE 2: ~-Galactosidase expression Plasmid Measurement p-Galactosidase activity No. (relative units, ODazo%mg min.) pAG-110 1 3.62 2 3.54 3 2.45 pAG-111 1 3.07 2 3.29 3 3.63 4 3.16 pAG-112 1 1.89 2 1.90 3 1.79 4 1.75 pAG-113 1 0 pAG-114 1 0 pAG-115 1 0 Sequence listing No. 1:
Sequence type: nucleotide Sequence length: 409 base pairs Strandedness: single strand r r~ ~;
- 17 - O.Z. 0050/41686 Topology: linear Molecule type: genomic DNA
Source: A. gossypii Properties: promoter region AAGCAAAAAT TACGGCTCCT CGCTGCAGAC CTGCGAGCAG GGAAACGCTC CCCTCAGCAG 2=~0 CACTGAGGTT CTTCTTTCAT ATACTTCCTT TTAAAATCTT GCTAGGATAC AGTTCTCACA.360 No. 2 Sequence of the 2.1 kb EcoRI/BamHI fragment with the open reading frame of the TEF gene 5' OdATfQ'CL~ G1CC,CrL~Ct~ ~GlC ,~'aTld~U, CGe'fbCGlf! TGCC~C1~C
crTerTCrrr c~rxrac~rc c.~rT~ errcQacca T~cac~rrcrc ~c~carc 130 140 1sa 1so 1, o lao C9sACdIAEA GSLA~PGt39 TlAOQddAdQ dtI~C~Tfl Al.'GTTG1CL-I vdl~iTTC:dI:
190 200 210 220 ?30 240 GTCG1CTCTC ~7ldCI'C~1C TICCdCeOCt C1C:~TQ AC~Gi'f1'G6 I~GT~i'.GaC
250 260 270 280 2~0 300 AIGbGJI~CCI TCGAGidGIT CC~C~d6CbC G~'OCCG1C! TGCCrAdf,GC Ti'C~'t2Cl~C
- 310 320 330 340 3:4 360 Tack rrrn~au u°rcaacas c~cac~cac~ c~ccr~TC~.e caTacacaTr xc~~rTa~ acrrarcac Ta~ucrx caccrc~crc Tc~TTCacx aracxcc~c uo 4va 4so 460 4~0 4ao ac~caa~a rcaa~cat r~tavcca acTrar~c cnac~vc casrrrcax 4~o soo slo 520 spa s4o ~rroabcrc ~czoccrca crr~cocr ~ra~rcr~ bccaCOCTCn s~cam~c sso sso s7o sao s9o soo caooc:~TCZ rcocrriac ct~ccci~c uoca"crsw x~rrcccar caacaar~Tc 6I0 620 630 6t0 6=0 660 Gl,CPGCC~CCd CTCf~Gi3~C GGCa6d~~ lCI~GGa6lC CeCCLiCrlC
IT~Cl~GC TCCC~C7d CCCTIaG~S 6~TGGCIT3eC TTCC11TCYC C~CfG~C
730 740 7so 7so 770 7ao - 18 - O.Z. 0050/41686 No. 2 (Continuation) 'w'1'CrlCdalC~1 TC~ll1'G~CGC C7~CC1CC~1C GCt~dTGC! ~1GCGCIG GwIG~GWC
190 E00 d14 d20 830 880 ACC.uGCCTG GTGCCCTC~ GGGTaIC~bCC 1RG1'T~GG CClIC11TC~CCCd 850 860 8?0 8E0 890 900 CCrCTCaGaC CuCTC71C3a CGGTTG16~ TfGCGT~'OC IGa GGTxTTGGEl CGQTCG6T CGGC71G1G!'C G1GJICCGCI'C TCbTCJ~GCC aCGT~TGQ'1' 970 9a0 990 1000 1010 1020 GiTIC~TTCC CCCL"11~C TAT G? CC~fC~IGIT GCa"CCaCCaG
1030 lOdO 1050 1060 1070 1080 ~11TIGCiCC aGO~'C1~C 1GC1~'dClbC G~OG°f~ Cll CT;~:CGTC
1090 1100 1110 11~ 1130 lld0 ~IGGlG~ C11161GG111 CCPTlOC6CT dC~llGi LC4CLL'~CC 6~lCOQ'~?
u:~o ueo u~g uav ux~ uuu GaCI~ITCI 1C'CCT1~Q' CaTTQ°f~l'C M~CCC~C G1d181~C1C 2GC~G1?1C
TCTCGGTC! TC~Ci'OC~C b!i'OC11GTD 1GT1'CCdCGa CT!'cT~GiC
1~0 ugp u9p 1300 1310 1320 J1IGMCGbCI GadG~iGEdCiT6 CiaG~C ClIllC1'TCC1' u,IAO~~°t C~dC4C'~Cl '~rGTCBdCT! 'fG~°OC~l~C 1M~CCi~T6? CTCinJGGC 'rPi~0.G~C
1390 1400 IdlO 140 1130 1410 ray r~r~c,~ct ccC~c~ G~cas~c ~eca~cc 1450 . 1160 1170 1110 Id90 1300 ~ICnTC TTGTCAiGTC CCaC81o0CT G~?~CCTC1 CCiBGOCCOC cc~6oQ
151A L'u0 1330 3510 1550 1580 GCt~t lGa6TB~CPC liC3ri1'J~il~ Gdn'C~TGY! Zl°ClICUCa 'TCTC~Ti1'Cd 1570 1u0 1594 1600 1610 1620 bTl~'! ?ITlin?u '1~'t~?d'li't i~3'G1~! CllaA0~1'Gi ?llI~R~S
1630 1610 1650 1564 16'0 1680 T'1TT~CTCG 1GITClTCTC CCCaGaTCCC ~GtTIIIGTG C~C~6T A1T~TGC
crcSaBTCet, T3t3AiTOC! uaxo~?a2d C.roetcTCO~1 xrCQix~,C:a eC90G9CGr 1750 1760 17?0 1780 1190 1800 CQGTC1~C'd8 a:I'G1'C~Aa2 TT(~CC1GCCT ClI~TGCCTC Ca6G~txlGldl T11'GCZCGaC
~Q'GT~ C AaGGITIiCC CaTa1'GC~CP ATCGGCGG<1G allICGPTGC
CACJIGOC~'rCT 1'CC'11~L~ GdCCTO~~CC ?TCCaCTGCl' IGITCICItiG T1CGCGr.TIG
'lTaaCTTTC C1GOCCI~'! ~TOOCGCa ITIsIJSOC1T aTP60Gt'i~ GCtaCGeCl2 1990 2000 2014 ~0~0 2034 1040 CTGIOOCaIGa CGlCCl ~GGaCD00N' iTi~Tl2~ GGDC1CGG4 2050 2060 2090 x030 2090 ~~1TI1'11W GCCaCCOaIC CaTGl'L'GCa? :C~Za? TG11CG~'dG GbTCC 3' 2(~~~~~
- 19 - O.Z. 0050/41686 No. 3 Sequence of the kanamxcin-resistance gene - TEF promoter region fusion (TEF promoter region sequences underlined) 20 30 ;0 50 50 5' CTCGaC'i'C2A ATC~JC C~idCCGAd CAtTCATGG PClaMAA2C c~ITCLTu 7o ao 90 loa 1.o zzo A4CCC1'fXbC AGCOCGC1GG Gf~d(~fGa ATACCCCP~! M2GICClaC ACdCTC~TGA
130 140 150 160 1'0 180 TG(xuCCTC 1GL121t~,bC TGICCfCI'OC Q'CGTGUGa AGC;"CTTCQ GBCPCbTACV
190 200 210 220 2;0 2a0 iGGCCfCUT CCCCC:ATG 'rCGGCGGa AaGTGaGG~ GCCICfCITC A.TCaGaGCIR
250 260 270 280 2~0 300 T~'."GP1GC! GG~ICCaGfTC fI'G31'i'Z'1'G~ A .. TGCCICG~i Ct~I'CTGCt?
'1'c~TCCwaaC l:GCCTGATC TGaT~I~Ca ~.TCac~ rICTTCGaITT ~Caa1 GtCdCCfTGT Gu;TCUUT f~7~Cfi AC~TfGGCa ACaTAaaAIT ATaTGTCIT
470 440 450 660 4i0 4E0 GLiCUTUI 1TACafluC~ G~dTICaaC GGG'1'QnT6 aOCCaTaTfC
AJiCs~G~llaC GTQTGQ'CC
:30 560 570 580 590 600 a 610 620 630 640 6:0 660 570 6a0 690 700 710 720 730 740 750 760 770 7a0 79o aoo slo ago a3a :4o aso aso a7o aao ago 900 GGCCCC6aT! laaTTOCaaC ~fCG~fiOCTC lTf1'lmTCO GTayuIIGC
970 980 990 1000 1010 10x0 i9 1TCTCG~C1 ATCdlCdaT~TC C1TTCT~C Cu~3 1030 1014 1050 1060 1070 10x0 cc~caracr xrrl~crcaa as cc~cccr~c craaTCxxr racacarAc Axcr~ca~ ~aucacocr caa~caara aZC~crrc c~am~a c~TZr~tc II50 1150 2170 ZZaO 1190 1200 CC?lt~'CCTG aT~'GC1TC GdT,CTC1CC ICfOOGi2CC OC~6aauC IGCaTTfC3IC
1210 1210 1230 1240 1250 12b0 CrllTaGiaG aaTaTOG'TCa lTnOr.~Gaa laTaTT4'11°0 a'1'GlQCtTCLTG
C~61TOC 1!'1'CGiI'rOC TGrIIlGTld! ~ aCl~ilrC Ca'IiTi°CG~
1330 1340 1350 136!1 I37A 13a0 Q'C~OC OOCUl~OC ui~'11C OCfiT~T~6 A~CWQra i~ITCfI~C
1390 1400 1~10 1~120 1430 1140 carp ccxoc~ra xaacucrc rcrsucua T~arucca ~c~rx s ua~aC gas rrcrcaax aruscxrar ~c 15!o lsao 133a is4o ipso ipso a tai xo ~~~c~ta r~cxcac~ ariCCaaar 1570 15E0 1590 1600 1610 lbZO
C'!t'GCClfCC TITC~ICl~C OC~TGIG lt~~T~'! CaTTIGGlI AC~T'iR°!
~~~'~~o - 20 - O.Z. 0050/41686 No. 3 (Continuation) 1630 16x0 1650 1660 16'0 1580 Cl~2bTC G:ITTGaTLI TC~ITI~TTC ldTIA~TTOC dG3T:~'.dT:T G~TGCTCGa2 GbGr~1TTC!' ~IITCaGadT? CCIT7IdTTOC TTGg3.lUQ' (~UGaGGI2 "3CCCTGACT
1750 1760 1770 1780 1,'90 1800 'fG~CGCG~CG GxGQ'tTGT TGa1T1,1TC GLICPrffCC I'G.1 .G':~d~lG C1TGG1TG
CCGTCTPCC C'~dCMCGCB C,~CCGTTC~ TG~GC~ dk~CTi~L ATCbCC.IdCT
GOTCC~CT1 GUC~GQ CTGTC~CC GTCOCTCCCT cad C~T&
G OIGCt~TTCa G~tGTl2 C~CTUt~ GC~PrZTTC ~CCBt~CdCA c~TUOCGC:T
aTrp'G~!' T~GTCaCG 1TC8Tt1~1CC CCTATrGGC Cl'GlCCCTGC
GCGCT~C1 G~ifir~iT C~TCc~ TI,C~CIWT G~~ITCflCCO TTGC~CC
CGGl!lLGG TxlC 3~
Xeys to Figures 1 to 8 Fig. 1: Plasmid pAG-1. ARS: S. cerevisiae ARS1 sequence;
2 micron; EcoRI fragment of the S, cerevisiae 2~
plasmid with replication origin; URA3:
S. cerevisiae URA3 gene; G418r: 6418 (kanamycin) resistance; black arrow: S. cerevisiae cycl-13 promoter; black box: S. cerivisiae CYC1 termin-ator; white arrows represent the direction of transcription.
Fig. 2: Plasmid pAG-2. amp: ampicillin resistance;
2 micron: EcoRI fragment of the S. cerevisiae 2~
plasmids with replication origin; URA3:
S. cerevisiae URA3 gene; G418r: 6418 (kanamycin) resistance; ORI: arigin of plasmid replication in E. coli; white arrows represent the direction of transcription.
Fig. 3: Plasmid pAG-100. amp: ampicillin resistance;
2 micron: EcoRI fragment of the S. cerevisiae 2~
plasmid with replication origin; URA3:
S. cerevisiae URA3 gene; G418r: 6418 (kanamycin) resistance; ORI: origin of plasmid replication in 2~c~.~'~r~~~
- 21 - O.Z. 0050/41686 E. coli; black arrow: A. gossypii DNA fragment with TEF promoter region; white arrows represent the direction of transcription.
Fig. 4: Plasmid pAG-5. amp: ampicillin resistance; G418r:
(kanamycin) resistance; ORI: origin of plasmid replication in E. coli; TEF: A. gossypii EcoRI/
BamHI fragment with ORF for the translation elongation factor; white arrows represent the direction of transcription.
Fig. 5: Plasmid pAG-101. amp: ampicillin resistance;
G418r: 6418 (kanamycin) resistance; ORI: origin of plasmid replication in E. coli; TEF:
A. gossypii EcoRI/BamHI fragment with ORF for the translation elongation factor; black arrow:
A. gossypii DNA fragment with TEF promoter-region; white arrows represent the direction of ' transcription.
Fig. 6: Plasmid pPLl. amp: ampicillin-resistance gene;
M13+: replication origin for single-stranded DNA
isolation; ori: origin for plasmid replication in E. coli; lacZ: E. coli lacZ gene; prom: 1500 by A. gossypii DNA fragment with the TEF promoter region.
Fig. 7: Plasmid pAG-110. 2u: EcoRI fragment of the S. cerevisiae 2~ plasmid with replication origin;
URA3: S. cerevisiae URA3 gene; prom: 1500 by A. gossypii DNA fragment with the TEF promoter region; lacZ: E. coli laxZ gene; G418r: 6418 (kanamycin) resistance gene; ari: origin for plasmid replication in E. coli; amp: ampicillin-resistance gene; black arrow: 1500 by A. gossypii DNA fragment with the TEF promoter region; white arrows represent the direction of transcription.
Fig. 8: Plasmid pAG-110. 2p: EcoRI fragment of the S. cerevisiae 2~ plasmid with replication origin;
URA3: S. cerevisiae URA3 gene; prom: 403 by A. gossypii DNA fragment with the TEF promoter 2~~~~~~
- 22 - O.Z. 0050/41686 region; lacZ: E. coli lacZ gene; G418r: 6418 (kanamycin) resistance gene; ori: origin for plasmid replication in E. coli; amp: ampicillin-resistance gene; black arrow: A. gossypii DNA
fragment with the TEF promoter region; white arrows represent the direction of transcription.
Fig. 9: Plasmid pPL2. amp: ampicillin-resistance gene;
M13+: replication origin foz single-stranded DNA
isolation; ori: origin for plasmid replication in E. coli; lacZ; E. coli lacZ gene; prom:
294 by A. gossypii DNA fragment with a part of the TEF promoter region (2?0 bp).
Fig. 10: Plasmid pPL3. amp: ampicillin-resistance gene;
M13+: replication origin for single-stranded DNA
isolation; ori: origin for plasmid replication--:
in E. coli; lacZ; E, coli lacZ gene; prom:.
239 by A. gossypii DNA fragment with a part of the TEF promoter region (215 bp).
Fig. 11: Plasmid pPL4. amp: ampicillin-resistance gene;
M13+: replication origin for single-stranded DNA
isolation; ori: origin for plasmid replication in E. coli; lacZ; E. coli lacZ gene; prom:
158 by A. gossypii DNA fragment with a part of the TEF promoter region (134 bp).
Fig. 12: Plasmid pAG-112. 2~: EcoRI fragment of the S. cerevisiae 2~ plasmid with replication origin; URA3: S. cerevisiae URA3 gene; prom:
294 by A. gossypii DNA fragment with the TEF
promoter region; lacZ: E. coli lacZ gene; G418r:
6418 (kanamycin) resistance gene; ori: origin for plasmid replication in E. coli; amp: ampi-cillin-resistance gene; black arrow: A. gossypii DNA fragment with the TEF promoter region; white arrows represent the direction of transcription.
Fig. 13: Plasmid pAG-113. 2~: EcoRI fragment of the S. cerevisiae 2;a plasmid with replication origin; URA3: S. c~revisiae URA3 gene; prom:
- 23 - O.Z. 0050/41686 239 by A. gossypii DNA fragment with the TEF
promoter region; lacZ: E. coli lacZ gene; G418r:
6418 (kanamycin) resistance gene; ori: origin for plasmid replication in E. coli; amp: ampi-cillin-resistance gene; black arrow: A. gossypii DNA fragment With the TEF promoter region; white arrows represent the direction of transcription.
Fig. 14: Plasmid pAG-114. 2~: EcoRI fragment of the S. cerevisiae 2~. plasmid with replication origin; URA3: S. cerevisiae URA3 gene; prom:
158 by A. gossypii DNA fragment with the TEF
promoter region; lacZ: E. coli lacZ gene; G418r:
6418 (kanamycin) resistance gene; ori: origin for plasmid replication in E. coli; amp: ampi-cillin-resistance gene; black arrow: A. gossypii-DNA fragment with the TEF promoter region; white ' arrows represent the direction of transcription.
FIg. 15: Plasmid pAG-115. 2~s: EcoRI fragment of the S. cerevisiae 2~ plasmid with replication origin; URA3: S. cerevisiae URA3 gene; lacZ:
E. coli lacZ gene; G418r: 6418 (kanamycin) resistance gene; ori: origin for plasmid repli cation .in E. coli; amp: ampicillin-resistance gene; white arrows represent the direction of transcription.
Fig. 16: TEF promoter fragments of the ,9-galactosidase expression plasmids.
Fig. I7: Nucleotide sequence in the ATG region and in the terminator region of MI3PT1, M13PT2, M13PT3, pAG-201, pAG-202, pAG-203.
Fig. 18: Plasmid pAG-201, pAG-202, pAG-203. 2~: ECORI
fragment of the S. cerevisiae 2~ plasmid with replication origin: prom, term: 751 by A. gossypii DNA fragment with the TEF promoter-terminator fusian. G418r:G418 (kanamycin) resistance gene; ori: origin point of plasmid replication in E. coli; amp:
~~, u~ ~; ~~ 'p. ~~ f.i - 24 - O.Z. 0050/41686 ampicillin-resistance gene; white arrows repre-sent the direction of transcription.
Fig. 19: Nucleotide sequence of the fusion of the promoter and terminator of the TEF gene.
Claims (7)
1. An isolated promoter of the translational elongation factor EF-1.alpha. gene of Ashbya gossypii comprising the nucleotide sequence as set forth in SEQ ID NO 1.
2. A genetically modified fungus into which the isolated promoter of claim 1 has been inserted.
3. Use of the genetically modified fungus of claim 2 for producing proteins.
4. Use according to claim 3 wherein the genetically modified fungus is Ashbya gossypii.
5. The use of the genetically modified fungus as claimed in claim 2 for an overexpression of genes of vitamin B2 biosynthesis or of genes connected with an overproduction of vitamin B2.
6. The use of the genetically modified fungus as claimed in claim 4 for an overexpression of genes of vitamin B2 biosynthesis or of genes connected with an overproduction of vitamin B2.
7. An isolated terminator of the translation elongation factor EF-1.alpha. gene of Ashbya gossypii comprising the sequence nucleotide 1513-2095 of SEQ ID NO 2.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4020181A DE4020181A1 (en) | 1990-06-25 | 1990-06-25 | NEW PROMOTERREGION |
| DEP4020181.3 | 1990-06-25 | ||
| PCT/EP1991/001116 WO1992000379A1 (en) | 1990-06-25 | 1991-06-15 | New promoter region |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2080482A1 CA2080482A1 (en) | 1991-12-26 |
| CA2080482C true CA2080482C (en) | 2002-10-01 |
Family
ID=6409025
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002080482A Expired - Lifetime CA2080482C (en) | 1990-06-25 | 1991-06-15 | Isolated promoter and terminator of elongation factor ef-1.alpha. |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0536192B1 (en) |
| JP (1) | JP3059214B2 (en) |
| AT (1) | ATE110779T1 (en) |
| CA (1) | CA2080482C (en) |
| DE (2) | DE4020181A1 (en) |
| DK (1) | DK0536192T3 (en) |
| ES (1) | ES2060392T3 (en) |
| WO (1) | WO1992000379A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5989870A (en) * | 1986-04-30 | 1999-11-23 | Rohm Enzyme Finland Oy | Method for cloning active promoters |
| CA2111709A1 (en) * | 1991-08-22 | 1993-03-04 | Sabine Steiner | The specific genetic modification of ashbya gossypii |
| DK0569806T3 (en) * | 1992-05-11 | 2001-12-31 | Basf Ag | DNA compounds and recombinant DNA expression vectors encoding riboflavin synthetase activity of S. cerevisiae |
| DE19521314A1 (en) * | 1995-06-12 | 1996-12-19 | Max Planck Gesellschaft | Adherence gene from Helicobacter pylori and polypeptide encoded thereby |
| EP0927761A3 (en) * | 1997-12-23 | 2001-09-05 | Basf Aktiengesellschaft | Purinebiosynthesis genes from Ashbya possypii and use for the microbial Riboflavinsynthesis |
| DE19801120A1 (en) | 1998-01-15 | 1999-07-22 | Basf Ag | New orotidine-5'-phosphate decarboxylase gene useful as selectable marker for microbial transformation |
| US7673432B2 (en) * | 2004-03-11 | 2010-03-09 | The Hong Kong Polytechnic University | Double-skin tubular structural members |
| CN108251476B (en) * | 2018-02-01 | 2021-09-07 | 湖南鸿鹰生物科技有限公司 | Method for extracting vitamin B12 from enzyme preparation wastewater |
-
1990
- 1990-06-25 DE DE4020181A patent/DE4020181A1/en not_active Withdrawn
-
1991
- 1991-06-15 DK DK91911248.2T patent/DK0536192T3/en active
- 1991-06-15 JP JP3510339A patent/JP3059214B2/en not_active Expired - Lifetime
- 1991-06-15 DE DE59102761T patent/DE59102761D1/en not_active Expired - Lifetime
- 1991-06-15 WO PCT/EP1991/001116 patent/WO1992000379A1/en active IP Right Grant
- 1991-06-15 AT AT91911248T patent/ATE110779T1/en not_active IP Right Cessation
- 1991-06-15 ES ES91911248T patent/ES2060392T3/en not_active Expired - Lifetime
- 1991-06-15 CA CA002080482A patent/CA2080482C/en not_active Expired - Lifetime
- 1991-06-15 EP EP91911248A patent/EP0536192B1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| EP0536192A1 (en) | 1993-04-14 |
| DE4020181A1 (en) | 1992-01-02 |
| DK0536192T3 (en) | 1994-10-03 |
| JP3059214B2 (en) | 2000-07-04 |
| DE59102761D1 (en) | 1994-10-06 |
| ES2060392T3 (en) | 1994-11-16 |
| WO1992000379A1 (en) | 1992-01-09 |
| ATE110779T1 (en) | 1994-09-15 |
| JPH06500007A (en) | 1994-01-06 |
| EP0536192B1 (en) | 1994-08-31 |
| CA2080482A1 (en) | 1991-12-26 |
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