CA2111709A1 - The specific genetic modification of ashbya gossypii - Google Patents

Abstract

A process is disclosed for purposefully modifying the genetic characteristics of Ashbya gossypii by homologous recombination.

Description

~Lll'7D9 o.z. 0050/42623 The specific genetic modification of Ashbya qossypii The present invention relates to a method for thespeci~ic genetic modification of Ashbya gossypii by homologous recombination, and to Ashbya gossypii strains modified by this method.
The filamentous hemiascomycete Ashbya gossypii is of great interest, for example, as riboflavin-producing microorganism.
A method for specific genetic modification by homologous recombination for the yeast Saccharomyces cerevisiae has been described by Struhl et al. tProc.
Natl. Acad. Sci. USA, 76 (1979) 1035-1039) and later extended by Rothstein (Meth. Enzymology Vol. 194, p.281 et seq.).
Homologous recombination has been observed in filamentous ascomycetes such as Aspergillus or Neurospora; howe~er, the proportion of homologous -~ recombination is often low compared with non-homologous recombination. In addition, reliable parameters leading preferentially or almost exclusively to homologous recombination are not known (Microbiological Reviews, 53 ;~ (1989) 148-170).
It is an object of the present in~ention to provide a~method for the specific genetic modification of ~,: : .
Ashbya gossyp1i by homologous recombination.
We have found that this object is achieved particularly effectively when Ashbya gossypii is tran~formed with vectors which contain the DNA intended for recombination flanked by one or moxe gene regions of Ash~ya gossypii. For the method according to the invention, preference is given to using as gene region of Ashbya gossypii DNA sequences in the region of the gene locus of the translation elongation factor EF-1~ (TEF
, ~
gene) The gene of the translation elongation factor EF-1~ (TEF) of Ashbya gossypii is con~ained in a 4.6 kb EcoRI-EcoRI fragment and a 6.3 kb BamHI-BamHI fragment , 2ii ~7~

2 - O.Z. 0050/42623 which can be isolated from Ashbya DNA gene banks by hybridization with a TEF probe from Saccharomyces cerevisiae (EM~O J.3, (1984) 3311-3315) (Fig. 12).
The coding part of the TEF gene is located within the overlapping 2.1 kb EcoRI-BamHI fragment whose sequence is shown in DNA Sequence Listing SEQ ID NO:l together with the adjacent sequences.
We have also found that the Ashbya gossypii strains modified by this method have valuable properties.
By transformation of Ashbya gossypii is meant the ~, ~ transfer of recombinant DNA into Ashbya gossypii nuclei ; or other DNA-bearing organelles by various methods.
, .
P~rticularly suitable transfer methods are protoplast transformation and~electroporation.
;~ 15 The protoplast transformation is expediently ;~ carried out by preparing protoplasts from the mycelium using enzymes~such ~as zymolase. The protoplasts are suspended in an aqueous calcium-containing buffer in a concentration of from l`to 10 x lOa/ml, preferably from 3 2;0 ~ to 5 x 10~/ml, and incubated with the purified DNA. The purified DNA is~added in an amount of from 1 to 50 ~g, preferably from 10 to 25 ~g, per 100 ~l of protoplast suspension. The~transformed Ashbya gossypii cells are ncubated in nutrient medium and streaked onto agar 25~ plates.~ When ~ a~ ;selection marker, for example an antibiotic-resistance gene, is used the successfully t}ansformed cells ca~be identified particularly easily by~ growth ~ on antibiotic-containing nutrient media.
Subsequent- clonaI purification via spore isolation is expedient.
Used as vectors for the transformation are recom-binant DNA constructs which contain the DNA intended for recombination ~lanked by one or more gene regions of Ashbya gossypii. A flanking gene region particularly suitable for homologous recombination is the TEF gene region. The DNA intended for recombination is, for ~; example, flanked as represented in the plasmids pAG-102 ~:

7 ~ ~
-~ - 3 - o.z. 0050/42623 and pAG-145 (Figs. 4 and 11).
The DNA for recombination can likewise originate from Ashbya gossypii; however, it is also possible to use DNA from other organisms, both of prokaryotic and of eukaryotic origin, or synthetic DNA.
When DNA from Ashbya gossypii is used as DNA for recombination, it is possible, for example, to place weakly expressed Ashbya gossypii genes under the control of a strong promoter (eg. TEF promoter) by integration which is base-pair accurate, and thus to increase expression without needing to use heterologous expression signals.
It is also possible with the method according to the invention for DNA from other organisms than Ashbya gossypii, for example from the genetically better defined yeast Saccharomyces cerevisiae or the bacteria sacillus subtilis or Escherichia coli, to be introduced specifically into Ashbya gossypii cells and to replicate stably and, where appropriate, to be expressed therein.
The DNA for recombination can be coding or non-coding DNA, and the use of coding DNA is preferred.
` The vectors used in the method according to the invention can, furthermore, contain other DNA sequences such as selection markers. Shuttle vectors which both replicate in bacteria and are used for the transformation of Ashbya gos~ypii are preferably used. For this purpose these vector~ usually have a bacterial DNA replication origin and one or more antibiotic-resistance genes. The plasmids pAG-102 and pAG-145 or derivatives thereof are par~icularly preferred as vectors (Figs. 4 and 11). By derivatives are meant those plasmids which contain other DNA sequences flanked by A. gossypii DN~ in addition to or in place of the G418-resistance-gene.
When circular DNA is used as vector, this is linearized, before the transformation of Ashbya gossypii, so as to result in fragments which ha~e a terminal A.
gossypii sequence and which initiate the homologous - 4 - o.z. 0050/42623 recombination.
The Ashbya gossypii strains which have been specifically modified by this method can be identified by the fact that integration of vector DNA h~s taken place at the predetermined sites of the homology region (eg.
TEF gene locus). This can be detected, for example, by Southern blotting with a vector probe. It is particularly straightforward to isolate modified Ashbya gossypii strains when the vector DNA contains a selection marker.
It is then possible to isolate exclusively the modified ~; strains under selective conditions, for example by :~ antibio~ic selection.
The strains genetically modified by homologous recombination with ~he plasmid pAG-102 are called LU8334 to LU8341. These strains have been deposited at the : Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, under the following numbers:
U 8334: DSM 6661 ` : LU 8335: DSM 6662 LU 8336: DSM 6663 :; :LU 8337: DSM 6664 LU 8338: DSM 6665 LU 8339: DSM 6666 : L~ 8340: DSM 6667 :` 25 :~ ~U:8341: DSM 6668 : The method according to the invention makes it : possible for specific genetic modifications, such as insertions, deletions and substitutions which are base-: pair accurate, to be effected straightforwardly and genetically stably~or unstably as desired. This means that the hemiascomycete Ashbya gossypii is available for : methods of génetic manipulation which aim at, for example, genetically stable overexpression through homologous promoters to an extent similar to the single-cell yeast S. cerevisiae.
: The following Examples illustrate the invention.

..

- 5 - o.Z. 0050/42623 Isolation of the Ashbya gossypii TEF gene region 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 hybridization 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 6.3 kb-long BamHI fragment hybridized with the hetero-logous TEF gene probe. Fragments with lengths in this range were eluted from agarose gels, cloned into the vector pUC8 (Vieira and Messing, Gene 19 (1982), 259-268) which has been cut with EcoRI and BamHI, 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), 3961-3~65) using the 32P-labeled heterologous probe. The positive clones contained either the 4.6 kb-long EcoRI fragment or the 6.3 kb-long BamHI
fragment. The two clones overlap in a region of 2.1 kb which harbors the homology with the TEF gene probe and which was sequenced. The sequence of the 2.i kb fragment and adjacent regions is ~ontained in SEQ ID NO: 1.
SEQ ID NO: 1 contains the open reading frame of 1377 bp (SEQ ID NO: 2), 436 bp of the 5' non-coding region and 686 bp of the 3' non-coding region. Subsequently the promoter region was isolated as 403 bp-long HindIII/
HincII fragment which, besides the 379 bp in front of the start codon, also harbors the first 24 bp of the open reading frame of the TEF gene, and was employed for pAG100 constructions.

Plasmid constructions a) The vector pAG-1 (Fig. 1) (deposited as DSM 6010), ~I~i7a~
- 6 - O.Z. 0~50/42623 a derivative of the vector pEX4, was prepared as described by Ernst and Chan, J.Bacteriol. 163 tl985), 8-14. pAG-l contains a 1.7 kb SalI fragment with the G418-resistance gene (G41~r), which codes for the aminoglycoside phosphotransferase (APH(3')I), of the transposon Tn903~ In the original pEX4 construct, initially the 1695 bp PvuII fragment of Tn903 (Oka et al., J.Mol.Biol. 147 (1981), 217-226) was ligated into a plasmid with filled-in SalI
cleavage sites. The SalI cleavage sites were retain~d during this, and the resistance gene can be isolated as 1.7 kb SalI fragment. pAG-l contains the Saccharomyces cerevisiae ARS elements ARSl and 2~
ARS and undergoes autonomous replication in Ashbya gossypii.

b) pAG-2 ~Fig. 2). The 1.7 kb SalI fragment with the G418-resistance gene was cut out of pAG-l and ~; inserted into the SalI cleavage site of the SO
cere~isiae-E. coli shuttle vector YEp24 (Botstein et al., Gene 8 (1979), 17-24; New England Biolabs Inc., Beverly, M~, USA, 1988-1989 Catalog, 112-113). The structure of the new plasmid - pAG-2 - was checked by restriction endonuclease mapping, using the XhoI
~leavage site located in the 1.7 kb SalI fragment to check the insert orientation. pAG-2 contains the Saccharomyces cerevisiae ARS element 2~ ARS and undergoes autonomous replication in Ashbya gossypii (Wright and Philippsen, Gene 109 (1991), 99-105).

;, c) pAG-100 (Fig. 3). A 403 bp long HindIII/HincII
fragment which contains the promoter region and the first 24 bp of the open reading frame of the gene for the translation elongation factor EF-l~ (TEF
gene) from A.gossypii was inserted into the XhoI
cleavage site of pAG-2, which is located 30 bp in the 3' direction behind the start of translation of , - 7 - o.z. ooso/42623 the G418-resistance gene, after the protruding ends had been filled in. The orientation of the fragment in the resulting plasmid pAG-100 was checked by restriction endonuclease mapping with HindIIIO
Insertion of the 403 bp-long fragment resulted in the 10 N-terminal amino acids of APH(3')I being replaced by the first 8 amino acids of the A.gossypii translation elongation factor EF~
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). pAG-100 contains the Saccharomyces cerevisiae ~RS element 2~ ARS and undergoes autonomous replication in Ashbya gossypii.

d) pAG-102 ~Fig. 4). The starting vector is pUC19 in which the BamHI site has been inactivated by cutt-ingr filling in and religation. The 4.6 kb EcoRI
fragment of the TEF region was cloned into the EcoRI
site of pUCl9 (arrow = open reading frame of the TEF
gene). Subsequently, the 2.1 kb SalI fragment from ~;~ p~G100 was, after the ends had been filled in, cloned into the SspI site (thin arrow = open reading frame of the G418-resistance gene; thick white arrow = TEF promoter HindI I I -HincII fragment).
;~ .
e) Constxuction of pAG-103 (Fig. 10) The 6.3 kb BamHI fragment with the TEF gene from A.
gossypii was cloned into the Bam~I cleavage site of the plasmid pBluescript II SK (Alting-Mees and Short, Nucl. Acids Res., 17 (1989) 9494). The orientation of this fragment was checked by restriction cleavages with EcoRI, HincII and HindIII
(TEF = TEF gene from A. gossypii, white arrow on black ground = TEF promoter).

,' D 9 - 8 - o.z. 0050/42623 f) Construction of pAG-121 A 669 bp SalI/HindIII fragment which harbors the 3' end of the G418-resistance gene was isolated from pAG-100 and inserted into the SalI/HindIII-cut plasmid pBluescript II SK + (Alting-Mees and Short, 1989).

g) Construction of pAG-122 A 940 bp fragment which harbors the completing 5' end of the G418-resistance gene fused to the TEF
promoter was inserted into the HindIII cleavage site of pAG-121. This fragment was obtained by cleavage of pAG-loO with HindIII. The insertion of this fragment results in a complete G418-resistance gene.

h) Construction of pAG-145 (Fig. 11) pAG-103 was partially cleaved with HindIII, and the linearized plasmid was isolated from an agarose gel.
The protruding ends were converted into blunt ends by filling in using the Klenow fragment of DNA-polymerase I, and a 1.64 kb BamHI/SalI-fragment from pAG-122 with the kanamycin-resistance gene under the control of the TEF promoter was in~erted after the protruding ends ~ad been filled in. The position and orientation of the G418-resistance gene was checked by ~estriction cleavages (white or shaded arrow on ~ black ground = TEF promoter (HindIII/HincII
fragment), shaded = G418-resistance gene). The fusion of the filled-in SalI and HindIII cleavage sites restores HindIII cleavage sites.

Transformation of A.gossypii with TEF gene region vectors The transformations were carried out as outlined below:

a~

- ~ - o.z. 0050/42623 inoculate 200 ml of MA2 with about 1-2x107 spores incubate in baffle flasks at 27C and 350 rpm for 32-40 h filter off mycelium 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 30C
in a shaker for 30 min suspend mycelium in 5-10 ml of SPEZ per g fresh weight incubate in a shaking waterbath at 30C, check for protoplast formation under the microscope (proto plast formation should have reached more than 90 after 30 min) filter protoplast suspension through glass ilter (Schott, porosity 1) centrifuge filtrate for 5 min (Sorvall SM24 rotor, 1800 rpm) wash sediment lx in 20 ml of ST and lx in 20 ml of STC
uspend protoplasts in 20 ml of STC and determine titer in a counter after cen~rifugation, resuspend protoplasts at a density of 4xlO~/ml in STC
add 100 ~l of protoplast suspension to DNA ln a maximum of 15 ~l of TE and mix (amounts of DNA for integrative transformat on with linearized TEF gene region vectors: 15-20 ~g) incub-ate at room temperature for 15 min cautiously add l ml of PTC40 and mix by inversion centrifuge for 5 min (Heraeus Biofuge A, 1500 rpm) carefully remove supernatant, and suspend sediment in l ml of SMTCI
incubate at 27~C for 3 h, mix by inversion about every 45 min after centrifugation, suspend sediments in 1 ml of SM
mix suspension with 9 ml of SMA2 top layer and add ~ i L i 7 3 ~
- 10 - O. Z . 0050/42623 ~o SMA2 plate (20 ml of SMA2 agar per plate) - incubate plates at 27C for 18 h - cover plates with G418 (0.54 ml of G418 stock solution + 0.46 ml of H2O + 6 ml of 0.5~ agarose (in H2O, preheated to 42C)) - incubate plates further at 27C, transformants are visible after 3-6 days.
Media and solutions Media: MA2: peptone (Gibco casein hydrolysate, No. 140) : 10 g/l yeast extract (Gibco) : 1 g/1 glucose 10 g/1 myo-inositol 0.3 g/1 SMA2 agar: sorbitol : 1 M
peptone : 10 g/l yeast extract : 1 g/l glucose 20 g/l myo-inositol 0.3 g/l agar (Gibco) 12 g/l : 20 SMA2 top layer: as SMA2 agar with 0.8% agarose in : place of agar Solutions: SD: lM sorbitol; 50 mM dithiothreitol :~ SPEZ: lM sorbitol; 10 mM Na phosphate buffer pH 5.8; 10 mM EDTA; 2 mg/ml Zymolyase ~:~ 25 20 T (Seikagaku Kogyo Co., Tokyo) ST: lM sorbitol; 10 mM tris-HC1 pH 8 STC: lM sorbitol; 10 mM tris-HCl pH 8;
10 mM CaCl2 TE: 10 mM tris-HCl; 1 mM EDTA
. EDTA: ethylenediaminetetraacetic acid Tris: tris(hydroxyethyl)aminomethane SDS: sodium lauryl sulfate ddH2O: double-distilled water TBE: 100 mM tris, 100 mM boric acid, 2 mM
EDTA, pH = 8.0 pTC40: 40% (w/v) polyethylene glycol 4000 (Merck); 10 mM tris-Cl pH 8; 10 mM

ù 9 ~ o z. 0050/42623 CaCl2 SMTCI: 50~ SM (see below); 50% STCi 0.03 g/l myo-inositol SM: 50~ 2M sorbitol; 50% MA2 S G418 stock solution: 20 mg/ml G418 (Geneticin, Gibco) in H2O

Transformation of A. gossypii with pAG-102 The plasmid pAG-102 (Figure 4) contains a 4.6 kb EcoRI fragment from the Ashbya gossypii TEF region (TEF
= gene of the translation elongation factor EF-1~. The plasmid also harbors a G418-resistance gene under the control of the TEF promoter, which confers resistance to the aminoglycoside G418 on Ashbya gossypii transformants.
The plasmid has no signals for autonomous replication in Ashbya gossypii. pAG-102 DNA was cut within the TEF
homolo~y region (3' end of the TEF gene) at the BamHI
ite and used for the transformation of Ashbya gossypii protoplasts as in Example 3. This allows homologous recombination 3' of the TEF gene to be induced. The eight independently obtained G418-resistant transformants (hU8334-LU8341, deposited at the DSM, Braunschweig) retained their G418 resistance after clonal purification and without selection pressure. The pAG-102 DN~ had been ~tably integrated into the Ashbya gossypii genome.
Figure 5a shows a chromosome fractionation of t~e eight transformants and of the wild-type strain (middle lane). The TEF gene is located on the largest of the five sible chromosomes, and the plasmid had also been integrated into this in all eight cases (Fig. 5b).
These integrations took place exclusively at the TEF locus, since the BglII fragment which contains the TEF locus is about 10 kb longer in all eight transform-ants than in the wild-type BglII fra~ment (Fig. 6).
Homologous recombination ought, as shown in Figure 7, result in a precise doubling of the 4.6 kb TEF region.
This is the case as is shown by analysis of the ~ t ~ V 3 - 12 - o.z. 0050/42623 transformants and wild-type DNA after cleavage with BamHI
and EcoRI (Fig. 8a,b).
To check, DNA frayments which con~ain the novel joins between chromosomal and plasmid DNA were cloned from three of the eight transformants. Sequence analysis in the region of the BamHI sites showed no change from the wild~type DNA.
After storage at -70 for one year, the eight transformants were reanalyzed after renewed mycelial 1~ growth and sporulation. This revealed evîdence of amplification since three of the analyzed clones harbored two copies of the plasmid, and two in fact haxbored three copies (Fig. 9), in some cases with sequence overlaps.
One of these overlaps probably led to tandem duplication lS of the G418-resistance gene. The reason for these overlaps is perhaps the presence of two TEF promoter regions per copy of the plasmid ~see Fig. 4).

Transformation of A. gossypii with pAG-145 A. gossypii protoplasts were transformed with BamHI-cut plasmid pAG-14S. This makes it po~sible simultaneously to induce homologous recombination 5' and

3' from the TEF gene. Four transformants were subjected to clonal purification and investigated by Southern analysis for integration of the kanamycin-resistance gene. The analyses of EcoRI-cleaved DNA were carried out wi~h two clonally purified strains of each ~ransformant, and the analyses of BglII-cleaved DNA were carried out with one ~lonally purified strain of each transformant.
The results of -these analyses are shown in Fig. 13 and Fig. 14, and interpretation of the data based on the model is carried out in Fig. 15.
All the transformants show two bands in the BglII
Southern: the larger band with a length of about 18.6 kb does not occur in the untransformed wild type. It corresponds to the TEF locus after integration of the G418-resistance gene (see Fig. 15b). The smaller band ~, - 13 - o.z. 0050/42623 with a length of about 17 kb also occurs in the wild type and corresponds to the TEF locus without integrated G418-resistance gene. It can be formed either by a wild-type copy of the TEF locus (see Fig. 15a), or by a secondary event after the integration, namely a deletion of the G418-resistance gene by homologous recombination between the two TEF promoter copies (see Fig. 15c).
This would be advantageous, for example, if the intention is to delete an integrated heterologous marker after integration of a sequence signal or of a gene.
The analysis with EcoRI-cleaved DNA was carried out in order to investigate this. The TEF promoter fragment of the Ç418-resistance gene is preceded by an additional EcoRI cleavage site which derives from the lS polylinker of pAG-122 and therefore it does not occur in front of the TEF promoter fragment of the TEF gene. If the 17 kb BglII fra~ment in the transformants had been ; produced by integration and subsequent deletion, the a~ditional EcoRI cleavage site ought to be detectable - 20 because it is located in front of the duplicated region and ough~ therefore not to be affected by the deletion (see Fig. lSc). In this case, the transformants should no ~onger have the 1.55 kb EcoRI fragment of the wild-type locus but, instead, a 1.32 kb fragment and a 0.27 kb fragment. The absence of the 1.55 kb fragment and the pre~ence of the 1.32 kb fragment were shown for transformants 1, 3, 4 and 5 ~Fig. 14).
A stability test was carried out with the clonally purified transformants 1 to 5. Mycelium was incubated on non-selective medium for six days and subsequently mycelium from the edge of each colony was transferred to a new plate of non-selective medium and to a plate containing selective medium. Ater incubation on non-selective medium for a further three days, mycelium was again transferred to new plates. After six days of non-selective growth, all the transformants were still G418-resistant. After nine days, transformants 1, 2 and ïd~
- 14 - O.Z. 0050/42623 3 had lost their resistance to G418. The Southern data indicate that the loss is caused by a homologous recombination between the TEF promoter fragments. The relatively high frequency of deletion of the heterologous marker (G418-resistance gene) observed with this construction is in some circumstances ad~-antageous for cotransformation.
a) Cultivation of Ashbya gossypii Cultivation in a liquid culture or on a plate is achieved by inoculation with mycelium or spores.
Medium: 1 liter MA-2: 1 g peptone 1 g yeast extract 10 g glucose 12 g agar Selective medium: MA-2-G418: MA-2 ~ 200 mg G418/ml b) DNA isolation 1. Make up liquid culture in MA-2 medium in a baffle flask. 200 ml of medium produce about 1.5 g of mycelium. Incubation at 27C for 48 h.
2. Wash mycelium: filter off mycelium with suc-tion take up mycelium in 30 ml of ddH2O
repeat 2-3 times 25 ~ 3. Then take up the mycelium (about 1.5 g) in 30 ml of SCE buffer SCE: 1 M sorbitol 0.1 M sodium citrate . 60 mM EDTA pH 8

4. Addition of 30 ml of 2-mercaptoethanol (14 M) , Addition of 5 mg of zymolase per 1.5 g of mycelium ~;~ 5. Incubation at 37C for 1 h 6. Check protoplast formation under the microscope and, if necessary, prolong incubation time 7. Addition of 3 ml of 0.5 M EDTA pH 7.5 Addition of 3 ml of 10% SDS

~11i 7~3 - 15 - o.z. 0050/42623 8. Incubation at room temperature for 5 min 9. Addition of 5 mg of proteinase K per 1.5 g of mycelium 10. Incubation at 37C for 1 h 11. Determination of the volume Addition of the same volume of 5M ammonium acetate solution 12. Centrifugation at 15,000 rpm for 15 min 13. Discard pellet 14. Ethanol precipitation with 2.5 times the volume 15. Centrifugation at 10,000 rpm for 10 min 16. Wash pellet in 70% ethanol, dry and resuspend in 4 ml of TE 10:1 17. Addition of 0.5 mg of RNAse 18. Incubation at 37C for 1 h 19. Phenol extraction 20. Phenol/chloroform extraction 21. Determination of the volume 22. DNA precipitation with the ~ame volume of 5M
ammonium acetate solution and 2.5 time3 the ~: volume of ethanol : 23. Resuspend DNA in 1 ml of TE 10:1.
c) DN~ cleavage for Southern analyses About 1 ~g of DNA were cleaved per mixture.
: 25 EcoRI/BamHI double digestion:
: DNA 20 ~1 , ~
10 x MS buffer 20 ~1 ddH2O 159 ~1 -BamHI 10 units 200 ~1 Incubation at 37C overnight Addition of 10 ~1 of AS buffer Addition of 10 units EcoRI
Incubation at 37C overnight BglII digestion:
DN~ 20 ~1 10 x MS buffer 20 ~1 6 - o.z. 0050/42623 ddH2O 159 ~1 BglII 10 units 200 ~l Incubation at 37C overnight Subsequently:
DNA precipitation with the same volume of 5M ammonium acetate solution and 2.5 times the volume of ethanol Centrifugation at 15,000 rpm for 15 min Wash pellet in 70~ ethanol Dry pellet and resuspend in 2Q ~l of TE 10:1.
Buffer: lxMS buffer: 50 mM NaCl 10 mM tris-HCl pH 7.5 - ' 10 mM MgCl 2 ~; 1 mM dithiothreitol AS buffer: 500 mM NaCl 400 mM tris-HCl pH 7.5 : Running conditions:
For Eco/Bam digestion: 0.8~ agarose gel 3 V/cm 8 h running time For BylII digestion: 0.6~ agarose gel : 1 V/cm 66 h running time The gel obtained in this way were transferred to 25 ~ Hybond N membranes by Southern transfer and baked at 80C for 2 h. Prehyb~idization, h~bridiza~ion and subsequent detection were carried out non-radioac-: ti~ely under stringent conditions ('Non-radioactive Label~ng and Detection' Applications Manual, Boehrin-~ ger Mannheiml Order Number: 1093 657).
~, d) OFAGE preparation 1. Determine fresh weight of the mycelium by filter-: ing with suction an MA-2 liquid culture 2. Resuspend 0.5 g of mycelium in 2 ml of 50 mM EDTA
pH 7.5 ~; 3. Addition of buffer I
: 4. Cautious addition of 5 ml of low melting agarose ~ iii7 ~3 7 - o.z. 0050/42623 (40C)

5. Place mixture in a Petri dish t5 cm diameter) and leave to solidify

6. Co~er with buffer II

7. Incubation at 37C overnight

8. Remove buffer II by aspiration and add buffer III

9. Incubation at 55C overnight

10. For preservation of the preparation, remove buffer III by aspiration and replace by 5 ml of 0.5 M EDTA pH 8.5, and store Petri dish at 4C.
Buffers: I: 3 mg of Zymolyase 320 ~1 of 0.5M dithiothreitol 0.9 ml of SCE
4.5 ml of 0.5M EDTA pH 8.5 50 ~l of lM tris-HCl pH 8 200 ~l of 0.5M dithiothreitol 250 ~l of ddH20 III: 5 mg of protein~se K
50 mg of N-lauroyl sarcosinate 4.5 ml of 0.5M EDTA pH 8 50 ~l of lM tris:HCl pH 8 450 ~l of ddH20 : Low melting agarose: 1~ in 0.125 M EDTA pH 7.5 e)~ OFAGE run (apparatus: Rotaphor , Biometra) : 1. Cut small block out of OFAGE preparation : 2. Wash 3 times for 1 h at room temperature in 5 ml ~: of 50 mM EDT~ pH 7.5 3. Place blocks in the pocket~ in the gel ; Gel: 1~ agarose gel in 0.4x TBE
4. Running conditions: 20 h running time ~, 48 ~ pulse duration - 4-9C buffer temperature 0.4x TBE running buffer 300 V voltage 80-90 mA current : Legends for Figures 5 to 9 and 12 to 14 Fig. 5 a: Chromosome separation wi~h OFAGE.

a 3 18 - O.Z. 0050/42623 From left to right: LU8334, LU8335, LU8336, LU8337, ATCC 10985, LU8338, LU8339, LU8340, LU8341.
b: Hybridization of the separated chromosomes with a pUCl9 DNA probe Fig. 6 Fractionation of BglII-cleaved DNA of the strains (from left to right) LU8334, LU8335, LU8335, LU8337, ATCC 10985, LU8338, LU8339, LU8340, ~U834~ and hybridization with a pAG-102 DNA probe : Fig. 7 Diagram of the integration of the plasmid ~: p~G-102 by homologous recombination. Chromosom-al DNA is indicated by thick lines, and the ~: plasmid DNA by thin or double lines.
Arrows show the position of the TEF reading frame. B=BamHI, E=EcoRI, Bgl=BglII
Fig. 8 a: Fractionation of BamHI plus EcoRI-cleaved pAG-102 and Ashbya gossypii DNA, and HindIII-and EcoRI-cut A DNA. From left to right: A, pAG-102, ATCC 10895, LU8334 to LU8341, A.
b: Hybridization with a pAG-102 DNA probe.
: c: Und~rexpo~ure of the pAG-102 lane.
Fig. 9: Analysis of BglII-cleaved DNA of the pAG-102 transformants 1 to 8 after double clonal ~ purification DNA of pAG-102 transformants after double clonal purification and of the untransformed ; wild-type was cleaved with BglII, fractionated ~:; on a 0.4% agarose g~.l and hybridized in a . Southern experiment with radio-labeled pAG-102 DNA. The fragment about 17 kb in size which corresponds to the wild-type copy occurred in none of the transformants. The bands which are present instead indicate single integration : 35 into transformants 2, 3 and 6 and double and ~ triple integrations into transformants 1, 4, 5, : 7 and 8, which is confirmed by further J
. ~ - 19 - o.z. 0050/42623 cleavages. Transformants 1 to 8 are derived from LU8334 to LU8341. By comparison with these strains, they have been clonally purified one further time (wt: wild ~ype). The length data are approximate.

Fig. 12 Comparison of the homologous regions used for the integration of pAG-102 and pAG-145.
a: Homologous region of pAG-1~2: 4.6 kb EcoRI
fragment b: Homologous region of pAG-145: 6.3 kb BamHI
fragment The arrow indicates the point in the genome in which the foreign DNA is integrated after homologous recombination.

Fig. 13: Southern with BglII-cleaved DNA from five pAG-145 transformants Genomic DNA from five clonally purified transformants of the plasmid pAG-145 and of the untransformed initial strain was cleaved wi~h BglII, fractionated on a 0.4~ agarose gel and hybridized in a Southern experiment with radio-labeled pAG-145 D~A. All the transformants (lanes 1 to 5), show a signal with a length of abou~ 17 kb which also occurs in the wild-type DN~ (lane 6~. In addition, all the ~ransformants possess a fragment wi~h a length of a~out 18.6 kb, which indicates integration of the G418-resistance gene into the 17 kb BglII fragment.

Fig. 14: Southern with EcoRI-cleaved DNA from five pAG-145 transformants Genomic DNA from in each case two clonally ^?~
.~ - 20 - o.z. 0050/42623 purified strains of five transformants of the plasmid pAG-145 and of the untransformed initial strain was cleaved with EcoRI, fractionated on a 0.8~ agarose gel and hybridized in a Southern experiment with radio-labeled pAG-103 DNA. Transformants 1 (lanes 1, 2)~ 3 (lanes 5, 6), 4 (lanes 7, 8) and 5 (lanes 9, 10) no longer have the 1.55 kb fragment which occurs in the wild-type DNA (11) but instead have a 1.32 kb fragment and, in addition, a 1.6 kb fragment which corresponds to the G418-resistance gene. Fragments of this length also occur in the lane with EcoRI-cleaved pAG-14S DNA ~lanes 12, 13).
Transformant 2 (lanes 3, 4) shows the 1.6 kb fragment (G418-resistance gene) and a 1.55 kb fragment, which indicates diploidization or a gene conversion. For this reason, OFAGE
(orthogonal field alternation gel electrophoresis), was used to investigate whether deploidization had taken place in respect of the largest chromosome on which the homolo~y region is located. No evidence was found of this. The presence of the 5.15 kb and ~ ~ 4.~ kb fragments and the absence of other signals prove the homologous recombination in ` ; all the transformants.

Fig. lS: -Model o~ the integration of the ~amHI fragment from pAG-145 into the genome of A. gos~ypii and of the subsequent deletion o the G418-resistance gene by homologous recombination.
Black bars: TEF promoter ~HindIII/HincII
fragment), G418r: G418-resistance gene, TEF: A.
gossypii TEF-gene, Arrows: direction of transcription.

O.~. 0050/42S23 ~ 9 .

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: BASF Aktiengesellschaft (B) STREET: Carl-Bosch-Strasse 38 (C) CITY: Ludwigshafen (E) COUNTRY: Bundesrepublik Deutschland (F) POSTAL CODE (ZIP): D-6700 (G) TELEPHONE: 0621/6048526 (H) TELEFAX: 0621/6043123 (I) TELEX: 1762175170 (ii) TIT~.E OF INVENTION: Verfahren zur gezielten genetischen Veraenderung von Ashbya gossypii : (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM:
Not Applicable (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER:
(2) INFORMATION FOR SEQ ID NO:l:
: ~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2496 base pairs (B) TYPE: nucleic acid (C) STRAN~EDNESS: single (D) TOPOLOGY: linear : ~ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Ashbya gossypii (ix) FEATURE:
(A) NAMEfKEY: CDS
~B) LOCATION: 437..1810 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
:: TGGGCGGCCC GCACTCGTGC CTTCTTCGCA CGGGCCGCCG AACCCCGCCA GGCCATCAAG 60 : CTTGCCTCGT CCCGCCGGGT CACCCGGCCA GCGACATGGA GGCCCAGAAT ACCCTCCTTG 120 Met Gly Lys Glu Lys Thr ~is Val Asn Val Val Val Ile Gly His Val Asp Ser Gly Lys Ser Thr Thr Thr Gly His Leu O.Z. 0050/42623 2ill7~3 Ile Tyr Lys Cys Gly Gly Xle Asp Lys Arg Thr Ile Glu Lys Phe Glu Lys Glu P~la Ala Glu Leu Gly Lys Gly Ser Phe Lys ryr Ala Trp Val Leu Asp Lys Leu Lys Ala Glu Arg Glu Arg Gly Ile Thr Ile Asp Ile Ala Leu Trp Lys Phe Glu Thr Pro Lys Tyr His Val Thr Val Ile Asp Pro Pro Gly His Arg Asp Ph~ Ile Lys Asn Met Ile Thr Gly Thr Ser Gln Ala Asp Cys Ala Ile Leu Ile Ile Ala Gly Gly Val Gly Glu Phe Glu Ala Gly Ile Ser Lys ASp Gly Gln Thr Arg Glu His ~la Leu Leu Ala Tyr Thr Leu Gly Val Lys Gln Leu Ile Val Ala Ile Asn Lys Met GAC TCC GTC AAG TGG GAC GAG TCC AGA TAC CAG GAG ATT GTC AAG GAG 94g Asp~ Ser Val Lys Trp Asp Glu Ser Arg Tyr Gln Glu Ile Val Lys Glu 160: 165 170 Thr Ser Asn Phe Ile Lys Lys Val Gly Tyr Asn Pro Lys Thr Val Pro Phe Val Pro Ile Ser Gly Trp Asn Gly Asp Asn Met Ile Glu Ala Thr A~C AAC GCC CC~ TGG TAC AAG GGC TGG GAG AAG GAG ACC AAG GCT GGT 1093 Thr Asn Ala Pro Trp Tyr Lys Gly Trp Glu Lys Glu Thr Lys Ala Gly Ala Val Lys Gly Lys Thr Leu Leu Glu Ala Ile Asp Ala Ile Glu Pro Pro Val Arg Pro Thr Asp Lys Ala Leu Arg Leu Pro Leu Gln Asp Val Tyr Lys Ile Gly Gly Ile Gly Thr Val Pro Val Gly Arg Val Glu Thr Gly Val Ile Lys Pro Gly Met Val Val Thr Phe Ala Pro Ser Gly Val O.~. 0~50/4~i623 3~ 3 :: 23 Thr Thr Glu Val Lys Ser Val Glu Met His His Glu Gln Leu Glu Glu ~85 290 295 Gly Val Pro Gly Asp Asn Val Gly Phe Asn Val Lys Asn Val Ser Val AAG GAG ATC AGA AGA GGT AAC GTT TGC GGT GAC TCC AAG AAC GAC CCA 142g Lys Glu Ile Arg Arg Gly Asn Val Cys Gly Asp Ser Lys Asn Asp Pro 320 3~i5 330 Pro Lys Ala Ala Glu Ser Phe Asn Ala Thr Val Ile Val Leu Asn His Pro Gly Gln Ile Ser Ala Gly Tyr Ser Pro Val Leu Asp Cys His Thr Ala His Ile Ala Cys Lys Phe Asp Glu Leu 1eu Glu Lys Asn Asp Arg Arg Thr Gly Lys Lys Leu Glu Asp Ser Pro Lys Phe Leu Lys Ala Gly Asp Ala Ala Met Val Lys Phe Val Pro Ser Lys Pro ~et Cys Val Glu Ala:Phe Thr Asp Tyr Pro Pro Leu Gly Arg Phe Ala Val Arg Asp Met 15 : 420 425 Arg Gln Thr Val Ala ~Val Gly Val Ile Lys Ser Val Val Lys Ser Asp 430 : 435 440 Lys~Ala Gly Lys Val Thr L~s Ala Ala Gln Lys Ala Gly Lys Lys 445~ 450 455 TAGAGTAACT GACAATA~AA AGATTCTTGT TTTCAAGAAC TTGTCATTTG TATAGTTTTT 1870 GACATCATCT GCCCAGATGC~GAAGTTAAGT GCGCAGAAAG TAATATCATG CGTCAATCGT 1990 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 458 amino acids -O.Z. 0050/4~623 ~ ~ ~ i 7 `~ .3 . 24 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SE~UENCE DESCRIPTION: SEQ ID NO:2:
Met Gly Lys Glu Lys Thr His Val Asn Val Val Val Ile Gly His Val 1 5 10 15sp Ser Gly Lys Ser Thr Thr Thr Gly His Leu Ile Tyr Lys Cys Gly Gly Ile Asp Lys Arg Thr Ile Glu Lys Phe Glu Lys Glu Ala Ala Glu Leu Gly Lys Gly Ser Phe Lys Tyr Ala Trp Val Leu Asp Lys Leu Lys Ala Glu Arg Glu Arg Gly Ile Thr Ile Asp Ile Ala Leu Trp Lys Phe 80lu Thr Pro Lys Tyr His Val Thr Val Ile Asp Pro Pro Gly His Arg 95sp Phe Ile Lys Asn Met Ile Thr Gly Thr Ser Gln Ala Asp Cys Ala Ile Leu Ile Ile Ala Gly Gly Val Gly Glu Phe Glu Ala Gly Ile Ser llS 120 125 1ys Asp Gly Gln Thr Arg Glu His Ala Leu Leu Ala Tyr Thr Leu Gly Val Lys Gln Leu Ile Val Ala Ile Asn Lys Met Asp Ser Val Lys Trp 145 150 155 160sp Glu Ser Arg Tyr Gln Glu Ile Val Lys Glu Thr Ser Asn Phe Ile 165 170 175ys Lys Val Gly Tyr Asn Pro Lys Thr Val Pro Phe Val Pro Ile Ser Gly Trp Asn Gly Asp Asn Met Ile Glu Ala Thr Thr ~sn Ala Pro Trp Tyr Lys Gly Trp Glu Lys Glu Thr Lys Ala Gly Ala Val Lys Gly Lys 210 ~15 220 Thr Leu Leu Glu Ala Ile Asp Ala Ile Glu Pro Pro Val Arg Pro Thr 225 230 235 240sp Lys Ala Leu Arg Leu Pro Leu Gln Asp Val Tyr Lys Ile Gly Gly 245 250 255'le Gly Thr Val Pro Val Gly Arg Val Glu Thr Gly Val Ile Lys Pro Gly Met Val Val Thr Phe Ala Pro Ser Gly Val Thr Thr Glu Val Lys Ser Val Glu Met His His Glu Gln 1eu Glu Glu Gly Val Pro Gly Asp Asn Val Gly Phe Asn Val Lys Asn Val Ser Val Lys Glu Ile Arg Arg 305 310 315 320ly Asn Val Gys Gly Asp Ser Lys Asn Asp Pro Pro Lys Ala Ala Glu 325 330 335er Phe Asn Ala Thr Val Ile Val Leu Asn His Pro Gly Gln Ile Ser O . Z . O O S O / 42 62 3 ~ i 1 i 7 a .. 25 Ala Gly Tyr Ser Pro Val Leu Asp Cys His Thr Ala His I le Ala Cys Lys Phe Asp Glu Leu Leu Glu Lys Asn Asp Arg Arg Thr Gly Lys Lys Leu Glu Asp Ser Pro Lys Phe Leu Lys Ala Gly Asp Ala Ala Met Val 385 390 395 400ys Phe Val Pro Ser Lys Pro Met Cys Val Glu Ala Phe Thr Asp Tyr 405 410 415ro Pro Leu Gly Arg Phe Ala Val Arg Asp Met Arg Gln Thr Val Ala 420 425 ~30 Val Gly Val Ile Lys Ser Val Val Lys Ser Asp Lys Ala Gly Lys Val Thr Lys Ala Ala Gln Lys Ala Gly Lys Lys .

Claims (6)

We claim:

1. A method for the specific genetic modification of Ashbya gossypii by homologous recombination, which comprises transforming Ashbya gossypii with vectors which contain the DNA intended for recombination flanked by one or more gene regions of Ashbya gossypii.

2. A method as claimed in claim 1, wherein DNA
sequences in the region of the gene locus of the translation elongation factor EF-1.alpha. are used as gene region of Ashbya gossypii.

3. A method as claimed in claim 1, wherein the plasmid pAG-102 or a derivative thereof is used as vector.

4. A method as claimed in claim 1, wherein the plasmid pAG-145 or a derivative thereof is used as vector.

5. A genetically modified Ashbya gossypii strain obtainable by a method as claimed in any of claims 1 to 4.

6. A genetically modified Ashbya gossypii strain selected from the group comprising LU8334 to LU8341.