GlaR (YugA)—a novel RpiR‐family transcription activator of the Leloir pathway of galactose utilization inLactococcus lactisIL1403

ABSTRACT Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.

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GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4991 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4991-4999 ◽

Cited By ~ 71

Author(s):

Y Suzuki ◽

Y Nogi ◽

A Abe ◽

T Fukasawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Sources ◽

Alpha Helix ◽

Normal Function ◽

Hybridization Experiment ◽

Yeast Cells ◽

Predicted Amino Acid Sequence ◽

Wild Type ◽

Transcription Activator ◽

Galactose Utilization

Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.

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Characterization, Expression, and Mutation of the Lactococcus lactis galPMKTE Genes, Involved in Galactose Utilization via the Leloir Pathway

Journal of Bacteriology ◽

10.1128/jb.185.3.870-878.2003 ◽

2003 ◽

Vol 185 (3) ◽

pp. 870-878 ◽

Cited By ~ 51

Author(s):

Benoît P. Grossiord ◽

Evert J. Luesink ◽

Elaine E. Vaughan ◽

Alain Arnaud ◽

Willem M. de Vos

Keyword(s):

Escherichia Coli ◽

Lactococcus Lactis ◽

Cell Separation ◽

Sole Carbon Source ◽

Wild Type ◽

Leloir Pathway ◽

Wild Type Phenotype ◽

Mutant Strains ◽

Galactose Utilization ◽

Long Chain

ABSTRACT A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose 1-epimerase (GalM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.

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Awakening the endogenous Leloir pathway for efficient galactose utilization by Yarrowia lipolytica

Biotechnology for Biofuels ◽

10.1186/s13068-015-0370-4 ◽

2015 ◽

Vol 8 (1) ◽

Cited By ~ 28

Author(s):

Zbigniew Lazar ◽

Heber Gamboa-Meléndez ◽

Anne-Marie Crutz- Le Coq ◽

Cécile Neuvéglise ◽

Jean-Marc Nicaud

Keyword(s):

Yarrowia Lipolytica ◽

Leloir Pathway ◽

Galactose Utilization

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GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4991-4999.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4991-4999

Author(s):

Y Suzuki ◽

Y Nogi ◽

A Abe ◽

T Fukasawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Sources ◽

Alpha Helix ◽

Normal Function ◽

Hybridization Experiment ◽

Yeast Cells ◽

Predicted Amino Acid Sequence ◽

Wild Type ◽

Transcription Activator ◽

Galactose Utilization

Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.

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Energetic limits to metabolic flexibility: responses of Saccharomyces cerevisiae to glucose–galactose transitions

Microbiology ◽

10.1099/mic.0.025775-0 ◽

2009 ◽

Vol 155 (4) ◽

pp. 1340-1350 ◽

Cited By ~ 33

Author(s):

J. van den Brink ◽

M. Akeroyd ◽

R. van der Hoeven ◽

J. T. Pronk ◽

J. H. de Winde ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Energy Charge ◽

Metabolic Flexibility ◽

Energy Status ◽

Anaerobic Conditions ◽

Leloir Pathway ◽

Chemostat Cultures ◽

Galactose Utilization ◽

Intracellular Energy ◽

Adenosine Nucleotide

Glucose is the favoured carbon source for Saccharomyces cerevisiae, and the Leloir pathway for galactose utilization is only induced in the presence of galactose during glucose-derepressed conditions. The goal of this study was to investigate the dynamics of glucose–galactose transitions. To this end, well-controlled, glucose-limited chemostat cultures were switched to galactose-excess conditions. Surprisingly, galactose was not consumed upon a switch to galactose excess under anaerobic conditions. However, the transcripts of the Leloir pathway were highly increased upon galactose excess under both aerobic and anaerobic conditions. Protein and enzyme-activity assays showed that impaired galactose consumption under anaerobiosis coincided with the absence of the Leloir-pathway proteins. Further results showed that absence of protein synthesis was not caused by glucose-mediated translation inhibition. Analysis of adenosine nucleotide pools revealed a fast decrease of the energy charge after the switch from glucose to galactose under anaerobic conditions. Similar results were obtained when glucose–galactose transitions were analysed under aerobic conditions with a respiratory-deficient strain. It is concluded that under fermentative conditions, the energy charge was too low to allow synthesis of the Leloir proteins. Hence, this study conclusively shows that the intracellular energy status is an important factor in the metabolic flexibility of S. cerevisiae upon changes in its environment.

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Unraveling the Leloir Pathway of Bifidobacterium bifidum: Significance of the Uridylyltransferases

Applied and Environmental Microbiology ◽

10.1128/aem.02460-13 ◽

2013 ◽

Vol 79 (22) ◽

pp. 7028-7035 ◽

Cited By ~ 15

Author(s):

Frederik De Bruyn ◽

Joeri Beauprez ◽

Jo Maertens ◽

Wim Soetaert ◽

Marjan De Mey

Keyword(s):

Escherichia Coli ◽

Human Milk ◽

Bifidobacterium Longum ◽

Bifidobacterium Bifidum ◽

Human Milk Oligosaccharides ◽

Content Type ◽

Leloir Pathway ◽

Functional Analog ◽

Major Route ◽

Galactose Utilization

ABSTRACTThe GNB/LNB (galacto-N-biose/lacto-N-biose) pathway plays a crucial role in bifidobacteria during growth on human milk or mucin from epithelial cells. It is thought to be the major route for galactose utilization inBifidobacterium longumas it is an energy-saving variant of the Leloir pathway. Both pathways are present inB. bifidum, and galactose 1-phosphate (gal1P) is considered to play a key role. Due to its toxic nature, gal1P is further converted into its activated UDP-sugar through the action of poorly characterized uridylyltransferases. In this study, three uridylyltransferases (galT1,galT2, andugpA) fromBifidobacterium bifidumwere cloned in anEscherichia colimutant and screened for activity on the key intermediate gal1P. GalT1 and GalT2 showed UDP-glucose-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.12), whereas UgpA showed promiscuous UTP-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.10). The activity of UgpA toward glucose 1-phosphate was about 33-fold higher than that toward gal1P. GalT1, as part of the bifidobacterial Leloir pathway, was about 357-fold more active than GalT2, the functional analog in the GNB/LNB pathway. These results suggest that GalT1 plays a more significant role than previously thought and predominates whenB. bifidumgrows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans.

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IMP1/imp1: A GENE INVOLVED IN THE NUCLEO-MITOCHONDRIAL CONTROL OF GALACTOSE FERMENTATION IN SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/97.1.27 ◽

1981 ◽

Vol 97 (1) ◽

pp. 27-44

Author(s):

A A Algeri ◽

L Bianchi ◽

A M Viola ◽

P P Puglisi ◽

N Marmiroli

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Gene ◽

Genetic Determinant ◽

Mitochondrial Inhibitors ◽

Leloir Pathway ◽

Induction Of Enzymes ◽

Gal Genes ◽

Galactose Utilization ◽

Respiratory Deficient

ABSTRACT In some strains of Saccharomyces cerevisiae, the induction of enzymes of the Leloir pathway, galactose fermentation and growth on galactose depend on mitochondrial function; mitochondrial dependence is elicited through the recessive allele imp1 of the nuclear gene IMP1. The genetic element IMP1 is not allelic to any of the known GAL genes; IMP1 strains can grow on and ferment galactose in respiratory-deficient (RD) condition or in the presence of the mitochondrial inhibitors ethidium bromide and erythromycin; whereas, imp1 strains can grow on and ferment galactose only in respiratory-sufficient (RS) condition. The imp1 elicited mitochondrial dependence apparently involves regulation of the synthesis of the galactose catabolizing enzymes and synthesis of the galactose specific permease. IMP1 is not the only genetic determinant that elicits an interaction of the mitochondrion and the expression of the Gal system; the GAL3 gene, whose role in galactose utilization is demonstrated by the long-term adaptation phenotype of gal3 RS mutants, gives rise to a noninducible phenotype in RD condition or in the presence of mitochondrial inhibitors.

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