Secretion of Kluyveromyces lactis Cu/Zn SOD: strategies for enhanced production.
The Kluyveromyces lactis Cu/Zn SOD gene (SOD1) was fused with the toxin K1 signal sequence to obtain extracellular production of superoxide dismutase. Kluyveromyces marxianus L3 and K. lactis MW98-8C strains were transformed and compared as hosts for the secretion. The effects of the media composition were evaluated: In K. lactis, the highest volumetric activity was obtained in YKK synthetic medium in the presence of Cu(2+)/Zn(2+) cofactors (9.6 kU l(-1)). In K. marxianus, active SOD was produced only in YPD medium supplemented with Cu(2+) and Zn(2+) (8.8 kU l(-1)). In order to improve the production of secreted active SOD in K. lactis, the SOD1 copper carrier (CCS1) was overexpressed and targeted to the secretory apparatus. A positive effect was observed only when K. lactis was grown in a medium without Cu(2+)/Zn(2+) supplement. The best performing culture conditions for K. lactis and K. marxianus recombinant strains were successfully applied to two laboratory-scale fed-batch processes, and volumetric SOD activities increased up to 19.4 and 24.1 kU l(-1), respectively.
Raimondi
Stefano
S
Department of Developmental and Cell Biology, University La Sapienza, Rome, Italy.
Uccelletti
Daniela
D
Amaretti
Alberto
A
Leonardi
Alan
A
Palleschi
Claudio
C
Rossi
Maddalena
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression; D007716:Kluyveromyces / Q000201:enzymology* / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D001426:Bacterial Proteins; D003067:Coenzymes; D003470:Culture Media; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc