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United States Patent (19) 11) Patent Number: 5,633,362 Nagarajan Et Al

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United States Patent (19) 11) Patent Number: 5,633,362 Nagarajan Et Al IIIUS005633362A United States Patent (19) 11) Patent Number: 5,633,362 Nagarajan et al. 45 Date of Patent: May 27, 1997 54 PRODUCTION OF 13-PROPANEDIOL FROM Daniel et al, FEMS Microbiol. Lett, 100, 281-286 (1992). GLYCEROL BY RECOMBINANT BACTERIA EXPRESSING RECOMBINANT DOL Forage, R.G. et al, Bacteriology, 149(2), 413-419 (1982). DEHYDRATASE Jeter, R.M., J. Gen. Microbiology, 136,887-896 (1990). 75) Inventors: Vasantha Nagarajan, Wilmington; Tong, I-T et al, Appl. and Environmental Microbiology, Charles E. Nakamura, Claymont, both 57(12), 3541-3546 (1991). of Del. Ichikawa et al. J. Ferment. Technol. 63(2), 135-141 (1985). 73 Assignee: E. I. Du Pont de Nemours and Sprenger, G.A. etal, J. Gen. Microbiology, 135, 1255-1262 Company, Wilmington, Del. (1989). Boenigk, R. et al, Appl. Microbiol, and Biotechnol, 38, (21) Appl. No.: 440,377 453-457 (1993). 22 Filed: May 12, 1995 Forsberg, C.W., Appl. Environ. Microbiol. 53(4), 639-643 (51) Int. Cl. ................ C07H 21/02; CO7H 21/04; (1987). v. C12N 1/21 Zeng, A-P. et al, Enzyme Microb. Technol., 15, 770-779 52 U.S. Cl. ....................... 536/23.1; 536/22.1; 536/24.3: (1993). 435/252.3; 435/252.33 58 Field of Search ................................. 536/22.1, 23.1, Bobik, T.A. etal, J. of Bacteriol., 174(7), 2253-2266 (1992). 536/24.3; 435/252.3, 252.33 Hartmanis, M.G.N. et al. Archives of Biochem. and Biophys ics, 245(1), 144-152 (1986). 56 References Cited Tobimatsu, T. et al., J. Biol. Chem., 270(13), 7142-7148 U.S. PATENT DOCUMENTS (1995). 4,935,554 6/1990 Murphy et al. ......................... 568/867 4,962,027 10/1990 Slininger et al. ... ...... 435/147 Primary Examiner-Stephanie W. Zitomer 5,015,789 5/1991 Arntz et al. ......... ... 568/862 Assistant Examiner-Jeffrey Fredman 5,164,309 11/1992 Gottschalk et al. ... 435/158 5,246,843 9/1993 Kasai et al. ............. .435/58 57 ABSTRACT 5,254,467 10/1993 Kretschmann et al. ................ 435/158 A process is provided for the bioconversion of glycerol to FOREIGN PATENT DOCUMENTS 1.3-propanediol in which genes from a bacteria known to WO93/25696 12/1993 WIPO ............................... C12P 7/18 possess a diol dehydratase enzyme for 1,2-propanediol deg radation are cloned into a bacterial host and the host is OTHER PUBLICATIONS grown in the presence of glycerol; expression of the foreign Gibco-BRL catalog (1993-1994) pp. 9-5. genes in the host cell facilitates the enzymatic conversion of Daniel et al., (Apr. 1995), "Purification of 1,3-propanediol glycerol to 1,3-propanediol which is isolated from the cul dehydrogenase from Citrobacter freundi and cloning, ture. sequencing and overexpression of the corresponding gene in Escherichia coli", J. Bacteriol. 177(8):2151-2156. 10 Claims, 4 Drawing Sheets U.S. Patent May 27, 1997 Sheet 1 of 4 5,633,362 FIG. pduC pduB pdu pduF pOCE <<o-CH-OH)-o FIG.2 Klebsiella ---->l MRSKRFEALAKRPVNQOGEWKEWIEEGFIAMESPNDPKPSIRIVNGAVTE 50 : Salmonella ---->1 MRSKRFEAIAKRPVNQDGFWKEWIEEGFIAMESPNDPKPSIKIVNGAVTE 50 51 LDGKPVEQFDLIDHEIARYGINLARAEEVMAMDSVKLANMLCDPNVKRSD 100 . : 51 IDGKPWSEF. 59 U.S. Patent May 27, 1997 Sheet 2 of 4 5,633,362 pduC Kp ----> 1 M. RSKRFEALAKRPVNQDGFvKEWIEEGFIAMESPNDPKPSIRIvNGAvr 49 ll. : : : . ::: : . dhoB Cf. --> 1 MRRSKRFEVLAQRPVNQDGLIGEWPEEGLIAMESPYDPAssvKVENGRIv 50 50 ELDGKPWEQFDLIDHFARYGINARAEEWMAMDSWKIANMLCDPNVKRS 99 . : : : : : : . : . 51 ELDGKSRAEFDMIDRFIADYAINVPEAERAMQLDALEIARMLVDIHVSRE 100 00 DIVPLTTAMTPAKIVEvvSHMNvvEMMMAMQKMRARRTPSQQAHVTNIKD l49 : : : : : : : ... : : . : 10 EIIAITTAITPAKRLEVMAQMNVVEMMMALQKMRARRTPSNOCHVTNLKD 150 150 NPvoIAADAAEGAWRGFDEQETTVAVARYAPFNAIALLVGsovgRPGVLT 99 : : . : : : . 151. NEvoIAADAAEAGIRGFSEQETVGIARYAPFNALALLVGSOCGAPGVLT 200 200 ocSLEEATELKIaightCYAETISvyGTEPviTDGDDiewskGFLAssy 249 : . : : ; . 201 ocSVEEATELEGRGITSYAETVSVYGTESvFTDGDDTPWSKAFLASAY 250 250 AsRGtkRFTSGSGSEVOMGYAEGKSMYLEARCIYITKAAGvoGLONGs 299 : . ; : . 251 ASRGLKMRYTSGTGSEALMGYSESKSMLYLESRCIFITKGAGvoGLONGA 300 F IG 3 300 vscIGVPSAVPsGIRAVIAENicSALDLECassNDOTFTHSDMRRTARL 349 O : . : 301 WSCIGMTGAVPSGIRAVIAENLIASMLDLEVASANDQTFSHSDIRRTART 350 350 LMoFLPGTDFISSGYSAVPNYDNMFAGSNEDAEDFDDYNVIQRDLKVDGG 399 : : ; . 35l LMQMLPGTDFIFSGYSAVPNYDNMFAGSNFDAEDFDDYNILQRDLMVDGG 400 400 LRPVREEDVIAiRNKAARALOAvfAGMGLPPrDEEVEAATyåHGSKDMP 449 : . : : . 401 LRPVTEEETIAIRNKAARAIQAVFRELGLPLISDEEVDAATYAHGSKDMP 450 A50 ERNIVEDIKFAoEINKNRNGivvKALAKGGFPDVAQDMLNiOKAKLTG 499 . : l l ; , ; ; : . : : : ; ; , ; ; 451 ARNVVEDIAAVEEMMKRNITGLDIWGAISSSGFEDASNILNMLRQRVTG 500 500 DYLETSAIIvGEGOVISAVNDviDYAGPATGYRLoGERNEErkNIPGALD 549 : : : : : . : : . : ... : : 501 DYLOTSAELDROFDVVSAVNDINDYQGPGTGYRISAERWAEIKNEAGVVQ 550 550 PNELG 555 s t . : : 551 PGSE 556 U.S. Patent May 27, 1997 Sheet 3 of 4 5,633,362 FIG. 4 P dhoB Kp. 1 . RAVIAENLIASMLDLEVASANDOTFSHSDIRRTAR 36 dhaE. Cf. ---> 301 WSCIGMTGAVPSGIRAVIAENLIASMLDLEVASANDQTFSHSDIRRTART 350 8 37 MQMLPGTDFIFSGYSAVPNYDNMFAGSNFDAEDFDDYNILORDLMVDGG 86 351 MQMLPGTDFIFSGYSAVPNYDNMFAGSNFDAEDFDDYNILQRDLMVDGG 400 87 LRPVTEAETIAIRQKAARAIQAVFREIGIPPIADEEVEAATYAQG. 3. : 4 Ol. LRPVTEEETIAIRNKAARAIQAVFRELGIPLISDEEVDAATYAHGSKDMP 450 U.S. Patent May 27, 1997 Sheet 4 of 4 5,633,362 car its its 5,633,362 1. 2 PRODUCTION OF 13-PROPANEDIOL FROM converted to DHA. It would be advantageous to convert all GLYCEROL BY RECOMBINANT BACTERIA the glycerol to 1,3-propanediol by expressing only the EXPRESSING RECOMBINANT DOL reductive pathway enzymes while providing a different DEHYDRATASE substrate for the generation of reductant. A preferred system would provide for a more efficient use of the glycerol FIELD OF INVENTION substrate while maintaining high yields of diol product. This invention relates to a process for the bioconversion It has long been known that a number of bacteria are of glycerol to 1,3-propanediol by recombinant bacteria har capable of using 1,2-propanediol is a sole carbon source. It boring a foreign gene encoding a diol dehydratase. is thought that this ability is conferred by a specific vitamin 10 B dependent diol dehydratase which is encoded by the pdu BACKGROUND operon. The pdu operon is linked to the cob operon which 13-Propanediol is a monomer having potential utility in encodes enzymes needed for the biosynthesis of vitamin B the production of polyester fibers and the manufacture of and both operons are subject to the regulation of the same polyurethanes and cyclic compounds. activator protein encoded by the c pock gene. A variety of chemical routes to 13-propanediol are 15 Recently the genes encoding the diol dehydratase of known. For example, 1.3-propanediol may be prepared from Klebsiella oxytoca were cloned and sequenced and the genes ethylene oxide and a catalyst in the presence of phosphine, were expressed in E. coli. Although active diol dehydratase water, carbon monoxide, hydrogen and an acid; by the was observed in these transformants, there is no evidence catalytic solution phase hydration of acrolein followed by that these clones are able to metabolize a carbon substrate to reduction; or from hydrocarbons such as glycerol, reacted in 1,3-propanediol. the presence of carbon monoxide and hydrogen over peri Various Salmonella and Klebsiella sp. are known to odic table group VIII catalysts. Although it is possible to produce a diol dehydratase which catalyzes the conversion generate 1,3-propanediol by these methods, they are expen of 1,2-propanediol, under anaerobic conditions, to propi sive and generate waste streams containing environmental onaldehyde and eventually to 1-propanol and propionic acid. pollutants. 25 The diol dehydratase has also been identified in Clostridia, Biological routes to 1,3-propanediol are known which and Propionibacterium but not in E. coli. The diol dehy utilize feedstocks produced from renewable resources. For dratase from Klebsiella sp. can convert glycerol to 13 example, bacterial strains able to convert glycerol into propanediol (Forage et al., Bacteriol, 149, 413 (1981)). 1,3-propanediol are found e.g., in the species Klebsiella, 30 Although the primary function of the pdu diol dehydratase Citrobacter, Clostridium, and Lactobacillus. In these is in the metabolism of 1.2-propanediol, applicants have bacteria, glycerol can enter either an oxidative or reductive discovered that the expression of K. pneumoniae diol dehy pathway. Oxidation of glycerol results in the conversion of dratase in E. coli will catalyze the conversion of glycerol to glycerol to dihydroxyacetone (DHA) by glycerol dehydro 1,3-propanediol. The recombinant bacteria expressing the genase and the DHA is phosphorylated by an adenosine 35 dial dehydratase pathway converts glycerol to the desired triphosphate (ATP) dependent kinase to yield dihydroxyac 1,3-propanediol product and is not dependent on a linked etone phosphate (DHAP) which enters the glycolytic path system as with the glycerol dehydratase system. Applicants way in the cell. Reduction of glycerol is accomplished by an have discovered that transformation of recombinant bacteria initial isomerization and dehydration catalyzed by glycerol with the pdu diol dehydratase genes from Klebsiella sp. dehydrates to yield 3-hydroxypropionaldehyde which is 40 affords a new, efficient and cost effective biological route to further reduced by 1,3-propanediol:NAD" oxidoreductase to 1,3-propanediol. yield 1,3-propanediol, a dead end cellular metabolite. The expression of at least the first two enzymes involved in the SUMMARY OF THE INVENTION oxidative pathway as well as the two enzymes involved in The present invention comprises a cosmid comprising a the reductive pathway in K. pneumoniae are coordinately
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