Shifting the fermentative/oxidative balance in Saccharomyces cerevisiae by transcriptional deregulation of Snf1 via overexpression of the upstream activating kinase Sak1p.
With the aim to reduce fermentation by-products and to promote respiratory metabolism by shifting the fermentative/oxidative balance, we evaluated the constitutive overexpression of the SAK1 and HAP4 genes in Saccharomyces cerevisiae. Sak1p is one of three kinases responsible for the phosphorylation, and thereby the activation, of the Snf1p complex, while Hap4p is the activator subunit of the Hap2/3/4/5 transcriptional complex. We compared the physiology of a SAK1-overexpressing strain with that of a strain overexpressing the HAP4 gene in wild-type and sdh2 deletion (respiratory-deficient) backgrounds. Both SAK1 and HAP4 overexpressions led to the upregulation of glucose-repressed genes and to reduced by-product formation rates (ethanol and glycerol). SAK1 overexpression had a greater impact on growth rates than did HAP4 overexpression. Elevated transcript levels of SAK1, but not HAP4, resulted in increased biomass yields in batch cultures grown on glucose (aerobic and excess glucose) as well as on nonfermentable carbon sources. SAK1 overexpression, but not the combined overexpression of SAK1 and HAP4 or the overexpression of HAP4 alone, restored growth on ethanol in an sdh2 deletion strain. In glucose-grown shake flask cultures, the sdh2 deletion strain with SAK1 and HAP4 overexpression produced succinic acid at a titer of 8.5 g liter(-1) and a yield of 0.26 mol (mol glucose)(-1) within 216 h. We here report for the first time that a constitutively high level of expression of SAK1 alleviates glucose repression and shifts the fermentative/oxidative balance under both glucose-repressed and -derepressed conditions.
Berlin University of Technology, Institute for Biotechnology, Department of Microbiology and Genetics, Gustav Meyer Allee 25, D-13355 Berlin, Germany.
Andreas M
AM
Raab
Verena
V
Hlavacek
Natalia
N
Bolotina
Christine
C
Lang
D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism; D005285:Fermentation / Q000235:genetics / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000235:genetics / Q000502:physiology; D010084:Oxidation-Reduction; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D023081:CCAAT-Binding Factor; C061437:HAP4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C071570:SNF1-related protein kinases; C094326:PRK1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases
Gene. 1985;33(1):103-19
2985470
Appl Microbiol Biotechnol. 1998 Jan;49(1):66-71
9487712
Appl Microbiol Biotechnol. 1995 Dec;44(1-2):147-56
8579828
J Biosci Bioeng. 1999;87(3):333-9
16232477
Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8839-43
12847291
J Biol Chem. 2003 Jul 11;278(28):26146-58
12676948
J Bacteriol. 2005 Feb;187(3):940-8
15659672
Gene. 1992 Sep 1;118(1):131-6
1511876
Science. 1997 Oct 24;278(5338):680-6
9381177
Microbiol Mol Biol Rev. 1998 Jun;62(2):334-61
9618445
EMBO Rep. 2004 May;5(5):532-7
15071495
FEMS Yeast Res. 2001 Jul;1(2):139-49
12702359
Biochem J. 2006 Feb 1;393(Pt 3):797-805
16201971
J Biosci Bioeng. 2000;90(6):619-24
16232921
Mol Cell Biol. 1984 Jan;4(1):54-60
6366513
Mol Cell Biol. 2000 Feb;20(4):1321-8
10648618
Genes Dev. 1996 Dec 15;10(24):3105-15
8985180
Mol Cell Biol. 2005 Mar;25(6):2138-46
15743812
Microbiol Mol Biol Rev. 2006 Mar;70(1):253-82
16524925
J Biol Chem. 1991 Nov 25;266(33):22199-205
1939242
Metab Eng. 2010 Nov;12(6):518-25
20854924
J Biol Chem. 2001 Sep 28;276(39):36460-6
11486005
Yeast. 1989 Jul-Aug;5(4):285-90
2528863
Mol Gen Genet. 1997 Sep;256(1):45-53
9341678
Biotechnol Lett. 2006 Oct;28(19):1601-13
16900335
FEMS Yeast Res. 2003 Oct;4(1):87-95
14554200
Appl Environ Microbiol. 2005 Oct;71(10):6185-92
16204537
J Biol Chem. 1983 Jan 25;258(2):1165-71
6337132
Eukaryot Cell. 2003 Apr;2(2):265-73
12684376
Microbiol Rev. 1990 Sep;54(3):211-25
2215420
Nucleic Acids Res. 1996 Jul 1;24(13):2519-24
8692690
Mol Cell Biol. 1989 Nov;9(11):5034-44
2557546
J Biol Chem. 2004 Sep 10;279(37):39165-74
15220335
Cell. 1982 Jan;28(1):145-54
7039847
Mol Cell Biol. 2003 Jun;23(11):3909-17
12748292
Int J Food Microbiol. 2010 Oct 15;143(3):150-60
20832886
Mol Biol Cell. 1997 Aug;8(8):1603-18
9285828
Curr Biol. 2003 Aug 5;13(15):1299-305
12906789
Genetics. 1984 Dec;108(4):845-58
6392017
Yeast. 1996 Mar 15;12(3):247-57
8904337
BMC Syst Biol. 2009 Feb 06;3:17
19200357
J Biosci Bioeng. 1999;87(1):28-36
16232421
Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3132-6
7724528
Mol Cell Biol. 1998 Nov;18(11):6273-80
9774644
J Biol Chem. 1990 Jun 25;265(18):10419-23
2191948
Proc Natl Acad Sci U S A. 1998 May 26;95(11):6245-50
9600950
Genome Biol. 2003;4(1):R3
12537548
Genetics. 1999 May;152(1):153-66
10224250
Microbiology (Reading). 2004 Apr;150(Pt 4):929-934
15073302
Appl Environ Microbiol. 2000 May;66(5):1970-3
10788368
Biochim Biophys Acta. 2002 Jan 17;1553(1-2):102-16
11803020
Mol Cell Biol. 2004 Sep;24(18):8255-63
15340085
Anal Biochem. 2003 Nov 1;322(1):134-8
14705791
Mol Cell Biol. 1996 Jun;16(6):2922-31
8649403
Mol Cell Biol. 1994 Mar;14(3):1979-85
8114729