Properties of the Hansenula polymorpha-derived constitutive GAP promoter, assessed using an HSA reporter gene.
The glyceraldehyde-3-phosphate dehydrogenase promoter, P(GAP), was employed to direct the constitutive expression of recombinant human serum albumin (HSA) in Hansenula polymorpha. A set of integration vectors containing the HSA cDNA under the control of P(GAP) was constructed and the elemental parameters affecting the expression of HSA from P(GAP) were analyzed. The presence of a 5'-untranslated region derived from the HSA cDNA and the integration of the expression vector into the GAP locus were shown to improve the expression of HSA under P(GAP). Glycerol supported a higher level of HSA expression from P(GAP) along with a higher cell density than either glucose or methanol. The growth at high glycerol concentrations up to 12% did not cause any significant repression of the cell growth. A high cell density culture, up to 83 g l(-1) dry cell weight with a HSA production of 550 mg l(-1), was obtained in less than 32 h of cultivation in a fed-batch fermentation employing intermittent feeding with 12% glycerol. The GAP promoter-based HSA expression system showed a higher specific production rate and required a much simpler fermentation process than the MOX promoter-based system, demonstrating that P(GAP) can be a practical alternative of the MOX promoter in the large-scale production of HSA from H. polymorpha.
Heo
Joo Hyung
JH
Korea Research Institute of Bioscience and Biotechnology, Yusong P.O. Box 115, 305-600, Taejon, South Korea.
Hong
Won Kyoung
WK
Cho
Eun Young
EY
Kim
Moo Woong
MW
Kim
Jeong Yoon
JY
Kim
Chul Ho
CH
Rhee
Sang Ki
SK
Kang
Hyun Ah
HA
D020121:5' Untranslated Regions / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005822:Genetic Vectors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D005990:Glycerol / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D012709:Serum Albumin / Q000096:biosynthesis* / Q000235:genetics*; D014170:Transformation, Genetic
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D012709:Serum Albumin; D000431:Ethanol; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005990:Glycerol