EP0354624A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
A method for identifying and using biosynthetic or regulatory genes for enhanced production of secondary metabolites
Assignee: DSM BV
•
Filing: 1989-08-09
•
Granted: —
•
Expires: 2009-08-09
Abstract
Abstract Novel methods and compositions are provided for the enhanced production of secondary metabolites. Methods are provided for identifying sequences which when transformed into secondary metabolite producing hosts, enhance the production of secondary metabolite. The process is exemplified for penicillin. In addition, the P. chrysogenum acyltransferase gene has been isolated and sequenced.
Inventors
- 👤 Martien A. M. Groenen
- 👤 Annemarie E. Veenstra
- 👤 Pieter Van Solingen
- 👤 Bertus Pieter Koekman
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 12:07
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1072; C07; C07K; C07K14/00; C07K14/37; C07K14/375; C12N15/11; C12N15/52; C12N15/63; C12N15/79; C12N15/80; C12N9/00; C12N9/10; C12N9/1025; C12N9/1029; C12N9/88; C12P; C12P37/00
Claims Analysis
Total Claims: 42
Claim 1
Independent Claim
115 words
A method for isolating DNA-fragments expressed during the metabolism of microorganisms, and which are directly or indirectly essential for the production of a secondary metabolite in this microorganism, preferably a bacterium or fungus, said method comprising: (a) screening a DNA library prepared from said microorganism with probes obtained from mRNA or DNA derived from a first culture of said microorganism, producing said secondary metabolite; (b) screening said DNA library with probes obtained from mRNA or DNA derived from a second culture of said microorganism or a mutant thereof, lacking the production of said secondary metabolite; and (c) identifying fragments comprising genes transcribed in said first culture which are not substantially transcribed in said second culture.
Claim 2
Independent Claim
115 words
A method for isolating DNA-fragments expressed during the metabolism of microorganisms, and which are directly or indirectly essential for the production of a secondary metabolite in this microorganism, preferably a bacterium or fungus, said method comprising: (a) screening a DNA library prepared from said microorganism with probes obtained from mRNA or DNA derived from a first culture of said microorganism, producing said secondary metabolite; (b) screening said DNA library with probes obtained from mRNA or DNA derived from a second culture of said microorganism or a mutant thereof, lacking the production of said secondary metabolite; and (c) identifying fragments comprising genes transcribed in said first culture which are not substantially transcribed in said second culture.
Claim 3
Dependent Claim
52 words
A method for preparing a DNA construct comprising: (a) isolating fragments expressed during the production of a secondary metabolite according to claim 1; and (b) subcloning the isolated fragments to form a DNA construct which contains the gene(s) of interest and all necessary transcriptional and translational elements and optionally a selection marker.
Claim 4
Dependent Claim
55 words
A method for obtaining or enhancing the production of a secondary metabolite in a microbial host comprising: (a) prepararing DNA constructs according to claim 3; (b) transforming a candidate host with these DNA constructs; (c) cloning the resulting transformants; and (d) identifying clones producing said secondary metabolite at a higher level than said candidate host.
Claim 5
Dependent Claim
19 words
A method according to Claim 1, wherein said first and second cultures have the ability to produce β-lactam antibiotics.
Claim 6
Dependent Claim
23 words
A method according to Claim 1, wherein said first and second cultures are Penicillium , Aspergillus , Acremonium , Flavobacterium or Actinomycetes .
Claim 7
Dependent Claim
87 words
A vector comprising a sequence encoding a gene directly or indirectly involved in the biosynthetic pathway leading to a secondary metabolite, optionally a marker for selection in a host producing said secondary metabolite and optionally a sequence for enhancing transformation efficiency of said vector in said host, wherein said gene is selected from the group consisting of the acyltransferase gene, cryptic gene Y, and the cryptic genes contained entirely or partly within the phages L12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16 and B23.
Claim 8
Dependent Claim
87 words
A vector comprising a sequence encoding a gene directly or indirectly involved in the biosynthetic pathway leading to a secondary metabolite, optionally a marker for selection in a host producing said secondary metabolite and optionally a sequence for enhancing transformation efficiency of said vector in said host, wherein said gene is selected from the group consisting of the acyltransferase gene, cryptic gene Y, and the cryptic genes contained entirely or partly within the phages L12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16 and B23.
Claim 9
Dependent Claim
15 words
A vector comprising a sequence encoding acyltransferase of a structure as given in Figure 3.
Claim 10
Dependent Claim
15 words
A vector comprising a sequence encoding acyltransferase of a structure as given in Figure 3.
Claim 11
Dependent Claim
39 words
A vector according to Claim 6, comprising a sequence encoding acyltransferase, which has been modified by insertion mutagenesis, deletion mutagenesis, a combination of insertion and deletion mutagenesis, or site specific mutagenesis of the DNA sequence specified in Figure 3.
Claim 12
Dependent Claim
17 words
A vector according to Claim 6, wherein said gene comprises another than the endogenous promoter for transcription.
Claim 13
Dependent Claim
14 words
A transformed host comprising a DNA construct prepared with the method of Claim 2.
Claim 14
Dependent Claim
86 words
A transformed host comprising as a result of transformation a copy of a sequence comprising a gene functional in said host and encoding a protein directly or indirectly involved in the biosynthetic pathway leading to a secondary metabolite resulting in increased production of said secondary metabolite, wherein said gene is selected from the group consisting of the acyltransferase gene, cryptic gene Y, and the cryptic genes contained entirely or partly within the phages L12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16 and B23.
Claim 15
Dependent Claim
86 words
A transformed host comprising as a result of transformation a copy of a sequence comprising a gene functional in said host and encoding a protein directly or indirectly involved in the biosynthetic pathway leading to a secondary metabolite resulting in increased production of said secondary metabolite, wherein said gene is selected from the group consisting of the acyltransferase gene, cryptic gene Y, and the cryptic genes contained entirely or partly within the phages L12, K9, C12, P3, K11, B13, B20, G3, G1, L10, K16 and B23.
Claim 16
Dependent Claim
34 words
A novel strain obtained by strain improvement procedures other than DNA mediated transformation using a transformed host comprising a vector according to Claim 6 or a transformed host according to Claim 10 or 11.
Claim 17
Dependent Claim
18 words
A transformed host according to Claim 10 or 11, wherein said host is capable of producing β-lactam antibiotics.
Claim 18
Dependent Claim
22 words
A transformed host according to Claim 10 or 11, whereinsaid host is a Penicillium, Aspergillus , Acremonium , Flavobacterium or Actinomycetes .
Claim 19
Dependent Claim
14 words
A transformed host according to Claim 10, wherein said secondary metabolite is a β-lactam.
Claim 20
Dependent Claim
12 words
A transformed host according to Claim 10, wherein said β-lactam is penicillin.
Claim 21
Dependent Claim
22 words
A transformed host according to Claim 14, wherein said host is the species Penicillium chrysogenum or Acremonium chrysogenum or E. coli .
Claim 22
Dependent Claim
25 words
A transformed host according to Claim 14, wherein said gene is under the transcriptional initiation regulation of other than the wild-type transcriptional initiation regulatory region.
Claim 23
Dependent Claim
28 words
A DNA construct comprising the P. chrysogenum phosphoglycerate kinase gene promoter and genes, prepared according to the method of Claim 2, under the transcriptional control of said promoter.
Claim 24
Dependent Claim
30 words
A DNA construct prepared according to Claim 2, wherein the selection marker is a phleomycin resistance gene, which is expressed under the transcriptional control of the phosphoglycerate kinase gene promoter.
Claim 25
Dependent Claim
19 words
A recombinant DNA construct comprising the promoter and translational activating sequence of the acyltransferase gene of Penicillium chrysogenum .
Claim 26
Dependent Claim
19 words
A recombinant DNA construct comprising the promoter and translational activating sequence of the acyltransferase gene of Penicillium chrysogenum .
Claim 27
Dependent Claim
16 words
A recombinant DNA construct comprising the 3′-regulatory sequences of the acyltransferase gene of Penicillium chrysogenum .
Claim 28
Dependent Claim
16 words
A recombinant DNA construct comprising the 3′-regulatory sequences of the acyltransferase gene of Penicillium chrysogenum .
Claim 29
Dependent Claim
24 words
A recombinant DNA construct that encodes acyltransferase activity and that can be identified by using a DNA compound of Figure 3 as a probe.
Claim 30
Dependent Claim
24 words
A recombinant DNA construct that encodes acyltransferase activity and that can be identified by using a DNA compound of Figure 3 as a probe.
Claim 31
Dependent Claim
14 words
A plasmid selected from the group consisting of PGJ01 A or B, or pRH05.
Claim 32
Dependent Claim
14 words
A plasmid selected from the group consisting of PGJ01 A or B, or pRH05.
Claim 33
Dependent Claim
21 words
A DNA construct comprising a transformation enhancing sequence comprising the sequence of Figure 4 joined to another than the wild-type sequence.
Claim 34
Dependent Claim
21 words
A DNA construct comprising a transformation enhancing sequence comprising the sequence of Figure 4 joined to another than the wild-type sequence.
Claim 35
Dependent Claim
22 words
A DNA sequence according to Claim 25, wherein said other than wild-type sequence comprises a gene encoding an enzymatic activity or Penicillium.
Claim 36
Dependent Claim
71 words
A method for providing improved yields of an antibiotic secondary metabolite comprising: (a) growing a transformed host comprising an extra copy of a sequence comprising a gene, isolated according to the method of Claim 1 or 2, encoding a protein directly or indirectly involved in the biosynthetic pathway of the production of an antibiotic secondary metabolite resulting in the enhanced production of said antibiotic; and (b) isolating the resulting antibiotic product.
Claim 37
Dependent Claim
12 words
A method according to Claim 27, wherein said antibiotic is a β-lactam.
Claim 38
Dependent Claim
20 words
A method according to Claim 28, wherein said host is a Penicillium Aspergillus , Acremonium , Flavobacterium or Actinomycetes .
Claim 39
Dependent Claim
13 words
A method according to Claim 29, wherein said host is Penicillium chrysogenum .
Claim 40
Dependent Claim
11 words
A method according to Claim 27, wherein said gene encodes acyltransferase.
Claim 41
Dependent Claim
20 words
A method according to Claim 27, wherein said gene encodes a protein of a structure as given in Fig. 3.
Claim 42
Dependent Claim
13 words
A method according to Claim 27, wherein said gene is cryptic gene Y.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.