Production of fungal alpha-amylase by Saccharomyces kluyveri in glucose-limited cultivations.
Heterologous protein production by the yeast Saccharomyces kluyveri was investigated under aerobic glucose-limited conditions. Alpha-amylase from Aspergillus oryzae was used as model protein and the gene was expressed from a S. cerevisiae 2 micro plasmid. For comparison, strains of both S. kluyveri and S. cerevisiae were transformed with the same plasmid, which led to secretion of active alpha-amylase in both cases. The S. cerevisiae 2 micro plasmid was found to be stable in S. kluyveri as evaluated by a constant alpha-amylase productivity in a continuous cultivation for more than 40 generations. S. kluyveri and S. cerevisiae secreted alpha-amylase with similar yields during continuous cultivations at dilution rates of 0.1 and 0.2 h(-1) (4.8-5.7 mg (g dry weight)(-1)). At a dilution rate of 0.3 h(-1) the metabolism of S. kluyveri was fully respiratory, whereas S. cerevisiae produced significant amounts of ethanol. A fed-batch cultivation was carried out with S. kluyveri where the biomass concentration reached 85 g l(-1) and the alpha-amylase concentration reached 320 mg l(-1). Even though S. kluyveri could be grown to high cell density, it was also observed that it has a high maintenance coefficient, which resulted in low biomass yields at the low specific growth rates prevailing towards the end of the fed-batch cultivation.
Møller
Kasper
K
Center for Microbial Biotechnology, BioCentrum-DTU, Building 223, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark. km@biocentrum.dtu.dk
Sharif
Mostafa Z
MZ
Olsson
Lisbeth
L
D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis; D012440:Saccharomyces / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics*
D011994:Recombinant Proteins; D000516:alpha-Amylases; D005947:Glucose