Summary
A hybrid cefE gene was constructed by juxtaposing promoter sequences from the Penicillium chrysogenum pcbC gene to the open reading frame of the Streptomyces clavuligerus cefE gene. In S. clavuligerus the cefE gene codes for the enzyme penicillin N expandase [also known as deacetoxycephalosporin C synthetase (DAOCS)]. To insert the hybrid cefE gene into P. chrysogenum the vector pPS65 was constructed; pPS65 contains the hybrid cefE gene and the Aspergillus nidulans amdS gene. The amdS gene encodes acetamidase and provides for dominant selection in P. chrysogenum. Protoplasts of P. chrysogenum were transformed with pPS65 and selected for the ability to grow on acetamide medium. Extracts of cells cultivated in penicillin production medium were analyzed for penicillin N expandase activity. Penicillin N expandase activity was detected in approximately one-third of the transformants tested. Transformants WG9-69C-01 and WG9-61L-03 had the highest specific activities of penicillin N expandase: 4.3% and 10.3%, respectively, relative to the amount of penicillin N expandase in S. clavuligerus. Untrasformed P. chrysogenum exhibited no penicillin N expandase activity. Analysis of the penicillin V titer revealed that WG9-61L-03 produced titers equal to that of the recipient strain while the amount of penicillin V produced in WG9-69C-01 was reduced by five fold.
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References
Abraham EP (1986) In: Queener SF, Webber JA, Queener SW (eds) Beta-lactam antibiotics for clinical use. Marcel Dekker Inc, New York, pp 103–116
Beri RK, Turner G (1987) Curr Genet 11:639–641
Brannon DR, Fukuda DS, Mabe JA, Huber FM, Whitney JG (1972) Antimicrob Agents Chemother 1:237–241
Bunnell CA, Luke WD, Perry FM Jr (1986) In: Queener SF, Webber JA, Queener SW (eds) Beta-lactam antibiotics for clinical use. Marcel Dekker Inc, New York, pp 255–284
Carr LG, Skatrud PL, Sheetz ME, Queener SW, Ingolia TD (1986) Gene 48:257–266
Dotzlaf JE, Yeh WK (1987) J Bacteriol 169:1611–1618
Dotzlaf JE, Yeh WK (1989) J Biol Chem 264:10219–10227
Hynes MJ, Corrick CM, King JA (1983) Mol Cell Biol 3:1430–1439
Kolar M, Punt PJ, van den Hondel CAMJJ, Schwab H (1988) Gene 62:127–134
Kovacevic S, Weigel BJ, Tobin MB, Ingolia TD, Miller JR (1989) J Bacteriol 171:754–760
Leskiw BK, Aharonowitz Y, Mevarech M, Wolfe S, Vining LC, Westlake DWS, Jensen SE (1988) Gene 57:187–196
Levinson A, Silver D, Seed B (1984) J Mol Appl Genet 2:507–51
Niedermayer AO, Russo-Aiesi SM, Lendzain CA, Kelly JM (1960) Anal Chem 32:664–666
Meselson M, Yuan R (1968) Nature 217:1110–1114
Queener S, Schwartz R (1979) In: Rose AH (ed) Secondary products of metabolism. Academic Press, London, pp 35–122
Queener SW, Ingolia TD, Skatrud PL, Chapman JL, Kaster KR (1985) In: Leive E (ed) Microbiology. American Society of Microbiology, Washington DC, pp 468–472
Queener SW, Wilkerson S, Tunin DR, McDermott JP, Chapman JL, Nash C, Platt C, Westpheling J (1984) In: Vandamme EJ (ed) Biotechnoogy of industrial antibiotics. Marcel Dekker Inc, New York, pp 141–170
Ramon D, Carramolino L, Patino C, Sanchez F, Penalva MA (1987) Gene 57:171–181
Samson SM, Belagage R, Blankenship DT, Chapman JL, Perry D, Skatrud PL, Van Frank RM, Abraham EP, Baldwin JE, Queener SW, Ingolia TD (1985) Nature 318:191–194
Samson SM, Dotzlaf JE, Slisz ML, Becker GW, Van Frank RM, Veal LE, Yeh WK, Miller JR, Queener SW, Ingolia TD (1987) Bio/Technology 5:1207–1214
Skatrud PL, Queener SW (1984) Curr Genet 8:155–163
Skatrud PL, Queener SW, Carr LG, Fisher DL (1987a) Curr Genet 12:337–348
Skatrud PL, Queener SW, Fisher DL, Chapman JL (1987b) Abstract for the SIM Annual Meeting. Baltimore, Maryland
Smith DJ, Bull JH, Edwards J, Turner G (1989) Mol Gen Genet 216:492–497
Veenstra AE, von Solingen P, Huininga-Muurling H, Koekman BP, Groenen MAM, Smaal EB, Kattevilder A, Alvarez E, Barredo JL, Martin J (1989) Proc 4th ASM Conf Genet and Molec Biol Ind Microrg. Am Soc Microbiol, Washington DC
Vieira J, Messing J (1982) Gene 19:259–268
Weigel BJ, Burgett SG, Chen VJ, Skatrud PL, Frolik CA, Queener SW, Ingolia TD (1988) J Bacteriol 170:3817–3826
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Cantwell, C.A., Beckmann, R.J., Dotzlaf, J.E. et al. Cloning and expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum . Curr Genet 17, 213–221 (1990). https://doi.org/10.1007/BF00312612
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DOI: https://doi.org/10.1007/BF00312612