Abstract
A gratuitous strain was developed by disrupting the GAL1 gene (galactokinase) of recombinant Saccharomyces cerevisiae harboring the antithrombotic hirudin gene in the chromosome under the control of the GAL10 promoter. A series of glucose-limited fed-batch cultures were carried out to examine the effects of glucose supply on hirudin expression in the gratuitous strain. Controlled feeding of glucose successfully supported both cell growth and hirudin expression in the gratuitous strain. The optimum fed-batch culture done by feeding glucose at a rate of 0.3 g h−1 produced a maximum hirudin concentration of 62.1 mg l−1, which corresponded to a 4.5-fold increase when compared with a simple batch culture done with the same strain.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Chaplin MF, Kennedy JF (1986) Carbohydrate analysis. IRL, Oxford
Hovland P, Flick J, Johnston M, Sclafani RA (1989) Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose. Gene 83:57–64
Huangpin BH, Da Silva NA (2000) Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis. Biotechnol Bioeng 67:408–416
Kabir MA, Khanday FA, Mehta DV, Bhat PJ (2000) Multiple copies of MRG19 suppress transcription of the GAL1 promoter in a GAL80-dependent manner in Saccharomyces cerevisiae. Mol Gen Genet 262:1113–1122
Kapat A, Jung JK, Park YH (2000) Effect of continuous feeding of galactose on the production of recombinant glucose oxidase using Saccharomyces cerevisiae. Bioprocess Eng 23:37–40
Kim MD, Rhee SK, Seo JH (2001) Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal δ-Integration. J Biotechnol 85:41–48
Klein CJL, Olsson L, Nielsen J (1998) Glucose control in Saccharomyces cerevisiae: the role of MIG1 in metabolic functions. Microbiology 144:13–24
Moreno F, Herrero P (2002) The hexokinase2-dependent glucose signal transduction pathway of Saccharomyces cerevisiae. FEMS Microbiol Rev 26:83–90
Napp SJ, Da Silva NA (1994) Enhanced productivity through gratuitous induction in recombinant yeast fermentations. Biotechnol Prog 10:125–128
Panuwatsuk W, Da Silva NA (2003) Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture. Biotechnol Bioeng 81:712–718
Platt A, Reece RJ (1998) The yeast galactose genetic switch is mediated by the formation of a Gal4p-Gal80p-Gal3p complex. EMBO J 17:4086–4091
Rao KJ, Kim CH, Chung BH, Sohn JH, Rhee SK (1998) Effect of galactose feeding on the improved production of hirudin in fed-batch cultures of recombinant Saccharomyces cerevisiae. Bioprocess Eng 19:385–388
Verrips T, Puboc P, Visser C, Sagt C (2000) From gene to product in yeast: production of fungal cutinase. Enzyme Microb Technol 26:812–818
Acknowledgements
We are grateful to Dr. Hyun-Ah Kang for her kind donation of a GAL1-disruption vector. This work was supported by the post-doctoral fellowship program of the Korea Science and Engineering Foundation (KOSEF) and the Ministry of Education, through the BK21 program.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kim, MD., Lee, TH., Lim, HK. et al. Production of antithrombotic hirudin in GAL1-disrupted Saccharomyces cerevisiae . Appl Microbiol Biotechnol 65, 259–262 (2004). https://doi.org/10.1007/s00253-004-1598-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-004-1598-2