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The Gal Operon of Streptomyces

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The Gal Operon of Streptomyces curopaiscnes raiemami European Patent Office @ Publication number: 0 235 112 Office europeen des brevets A2 EUROPEAN PATENT APPLICATION (21; Application number: 87870026.9 Qn) Int. CI.3: C 12 N 15/00 C 12 N 1/20 © Date of filing: 26.02.87 © Priority: 28.02.86 US 834706 © Applicant: SMITHKUNE BECKMAN CORPORATION 30.01.87 US 9419 One Franklin Plaza PO Box 7929 Philadelphia Pennsylvania 19103(US) 43) Date of publication of application: © Inventor: Adams, Craig W. 02.09.87 Bulletin 87/36 1773 South Buena Vista Corona California 92720(US) m) Designated Contracting States: AT BE CH DE ES FR GB GR IT LI LU NL SE @ Inventor: Fornwald, James Allan 104 Burnside Avenue Norristown Pennsylvania 19403(US) © Inventor: Brawner, Mary Ellen 126 Valley Stream Circle Wayne Pennsylvania 19087(US) © Inventor: Schmidt, Francis John 1404 Doris Drive Columbia Missouri 65201 (US) © Representative: Tasset, Gerard SMITHKLINE - RIT rue de I'lnstitut, 89 B-1330Rixensart(BE) 64) The gal operon of streptomyces. © A recombinant DNA molecule comprising the Strepto- myces gal operon galK gene; galE gene; p^Tgene; P1 prom- oter; P2 promoter; P2 promoter expression unit; P1 promoter regulated region; or the entire Streptomyces gal operon is prepared. t 4 m J jyuuii rummy uompany LIQ 0235112 -l- TITLE THE GAL OPERON OF 10 — STREPTOMYCES CROSS REFERENCE TO RELATED APPLICATIONS . This application is a continuation-in-part of 15 • v " ' Serial Number 834,706, filed February 28, 1986, which is pending. BACKGROUND OF THE INVENTION 20 This invention relates to a recombinant DNA molecule comprising the Streptomyces gal operon. Hodgson, J. Gen. Micro., 128, 2417-2430 (1982), report that Streptomyces coelicolor A3(2) has a glucose repression system which allows repression at the level of 25 transcription of the arabinose uptake system, one of the glycerol uptake systems, and also repression of the galactose uptake system in wild type strains. There is no report in Hodgson of actual galactose metabolism by S. coelicolor A3 (2).' 30 Okeda et al. MoT. Gen. Genet., 196, 501-507 (1984), report that glucose kinase activity, 2-deoxyglu- cose-sensitivity, glucose utilization and glucose repression were all restored to S_^ coelicolor A3(2) glk (glucose kinase) mutants transformed by a 3.5 kb DNA 35 fragment which contained the glk gene cloned from S . into coelicolor a phage . vector . Seno et al., Mol. Gen, lienec, the glycerol (gyl) operon of Streptomyces (1984) , report substrate- coelicolor, and state that such operon is inducible and catabolite-repressible. 1841-1853 Debouck et al., Nuc. Acids. Res., 13(6), the of coli consists of (1985) , report that g_al operon which specify the three structurally contiguous genes metabolism of galactose, i.e., enzymes required for the gaJLT qalE (uridine diphosphogalactose-4-epimerase) , 0 and (galactose-l-phosphate uridyltransf erase) £a_lK from a (galactokinase); that such genes are expressed that the polycistronic mRNA in the order E, T, K; of the expression of the promoter distal gene operon, to the g_a_lT qalK, is known to be coupled translationally ■5 it; that such translational gene immediately preceding between the end coupling results from a structural overlap ribosome binding of the galT coding sequence and the translational coupling of region of galK; and that the of these qalT and galK ensures the coordinate expression -° metabolism of " galactose. genes during the SUMMARY OF THE INVENTION This invention relates to a recombinant DNA molecule comprising a Streptomyces g_al operon £alK gene; expression unit, or P2 qalE gene; galT gene; P2 promoter derivative thereof as well as a 25 promoter or any functional recombinant DNA molecule comprising a Streptomyces £al regulated region or the operon PI promoter, PI promoter functional entire gal operon or any regulatable and derivative thereof. 30 This invention also relates to a recombinant DNA molecule comprising the Streptomyces g_al operon or any thereof and a regulatable and functional derivative functional DNA molecule operatively linked to such operon; and such DNA molecule, a recombinant DNA vector comprising replicon; a 35 znd, optionally, additionally comprising a with such method of preparing a host cell transformed - 3 - 02351 12 vector; the transformed host prepared by such method; a method of expressing such functional DNA sequence which comprises cultivating such transformed host under suitable conditions such that the functional DNA sequence is expressed; and to a method of regulating the expression of such functional DNA sequence which comprises cultivating such transformed host under conditions which regulate such expression. This invention also relates to a recombinant DNA 10 molecule comprising the Streptomyces gal operon P2 promoter expression unit or any functional derivative thereof and a functional DNA molecule operatively linked to such unit; a recombinant DNA vector comprising such DNA molecule, and, optionally, additionally comprising a ^15 replicon; a method of preparing a host cell transformed with such vector; the transformed host prepared by such method; and to a method of expressing such functional DNA sequence which comprises cultivating such transformed host under suitable conditions such that the functional DNA 20 • sequence is expressed.j This invention also relates to a recombinant DNA molecule comprising the Streptomyces gal operon PI promoter regulated region or any regulatable and functional derivative thereof and a functional DNA 25 molecule operatively linked to such region; a recombinant DNA vector comprising such DNA molecule, and, optionally, additionally comprising a replicon; a method of preparing a host cell transformed with such vector; the transformed host prepared by such method; a method of expressing such 3° functional DNA sequence which comprises cultivating such transformed host under suitable conditions such that the functional DNA sequence is expressed; and to a method of regulating the expression of such functional DNA sequence which comprises cultivating such transformed host under 35 conditions which regulate such expression. This invention also relates to a recombinant DNA lecule comprisiny t«c ^'^w..., — - and functional derivative omoter or any regulatable DNA molecule operatively ereof and a foreign functional recombinant DNA vector comprising nked to such region; a additionally :ch DNA molecule, and, optionally, method of preparing a host cell uprising a replicon; a the transformed host ransformed with such vector; method of expressing such -epared by such method; a comprises cultivating such actional DNA sequence which conditions such that the ransformed host under suitable and to a method of actional DNA sequence is expressed; of such functional DNA sequence egulating the expression such transformed host under hich comprises cultivating expression. onditions which regulate such recombinant DNA This invention also relates to a g_al operon P2 olecule comprising the Streptomyces derivative thereof and a romoter or any functional molecule operatively linked to such oreign functional DNA vector comprising such DNA egion; a recombinant DNA additionally comprising a lolecule, and, optionally, a host cell transformed -eplicon; a method of preparing transformed host prepared by such ,ith such vector; the such functional DNA nethod; and to a method of expressing cultivating such transformed host sequence which comprises such that the functional DNA ander suitable conditions sequence is expressed. method of This invention also relates to a utilizing host microorganism or enabling a non-galactose which comprises transforming cell to utilize galactose DNA molecule comprising a such host with a recombinant portion of the Streptomyces S^re^myces gal operon or any is functional derivative thereof, which gal operon, or any transformed host to utilize adequate to enable such also relates to the recombinant galactose, This invention method and to the host DNA vector employed in such prepared by such method. BRIEF DtibtKJ.fi iutM ur xrir. uimniimo Figure 1 represents a restriction endonuclease lividans 1326 galactose (sal) ap of the Streptomyces locations for structural peron and indicates approximate enes and promoters within the operon. Figure 2 represents a restriction endonuclease iap of plasmid pK21. Figure 3 represents a comparison of the estriction endonuclease maps of the S. lividans gal containing the S. peron and a restriction fragment :oelicolor galK gene. DETAILED DESCRIPTION OF THE INVENTION It has now been discovered that the Streptomyces the metabolism of galactose jenome contains a operon for structural [i.e., a gal operon) which comprises three and two (PI and jenes (galT, galE and galK) promoters '2). The galT gene product is known as galactose-l- ,' the phosphate uridyltransf erase ( transferase) galE gene product is known as uridine diphosphogalactose- is 1-epimerase (epimerase) , and the galK gene product The function tnown as galactose-l-kinase (galactokinase) . and in Df the gene products of galT, galE g_alK galactose netabolism in Streptomyces is explained by the following diagram: 1. galactose + ATP galactokinase galactose-l-phosphate + ADP 2. galactose-l-phosphate + UDP-glucose transferase UDP-galactose + glucose-l-phosphate 3. UDP-galactose epimerase UDP-glucose By the term "promoter" is meant any region binding of RNA upstream of a structural gene which permits polymerase and transcription to occur. gy tile LC1IU oliui-i.---- which serves to be the template sequence for a polypeptide of mRNA. for the synthesis
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