Acetolysis and 1H NMR studies on mannans isolated from very flocculent and weakly flocculent cells of Pichia pastoris IFP 206

Mbawala, Augustin; Mahmood, S.; Loppinet, V.; Bonaly, R.

doi:10.1099/00221287-136-7-1279

Cited by 18 publications

(11 citation statements)

References 16 publications

(19 reference statements)

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“…Cell-wall mannan content was originally implicated in flocculation some 30 years ago (Devreux, 1962;Masschelein et al, 1963;Windisch, 1968). Flocculent cell-walls of S. cerevisiae have been shown to contain more mannan (Stewart and Garrison, 1972;Stewart, 1975), as has also been reported in Kluyveromyces bulgaricus (Al-Mahmood et al, 1987) and Pichia pastoris (Mbawala et al, 1990). Nishihara and Toraya (1 987) demonstrated yeast strains lacking flocculation receptors, until chemical treatment exposed receptors in these strains.…”

Section: Introductionmentioning

confidence: 79%

Yeast flocculation: Flocculation onset and receptor availability

Stratford

1993

Yeast

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Flocculent strains of brewing yeast grow and ferment as single cells and flocculate in the stationary phase of growth. The switch from single-celled yeast growth to multi-celled aggregation, flocculation onset, is of critical importance to the brewing industry. Yeast flocculation involves adhesion of surface-lectins on flocculent cells to carbohydrate receptors on neighbouring cell-walls. The presence of carbohydrate receptors, outer-chain mannan side-branches, was monitored throughout growth of flocculent and non-flocculent strains of Saccharomyces cerevisiae, with particular attention to the growth phases where flocculation is normally developed. Receptors were probed by coflocculation with flocculent strains and by aggregation with concanavalin A, a lectin shown to use the same receptors as flocculation. While considerable variation was found in coflocculation and concanavalin A aggregation between strains, little or no change in receptor availability was found throughout the growth of all yeast strains. Yeast cells could easily be coflocculated at any growth stage. It was concluded that receptor availability is not involved in the process of flocculation onset.

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Section: Introductionmentioning

confidence: 79%

Yeast flocculation: Flocculation onset and receptor availability

Stratford

1993

Yeast

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“…The data examined here exclusively concern flocculation of strains of S. cerevisiae. While flocculation has recently been demonstrated in several other yeast genera: Hansenula anomala (Moriya et al, 1990;Saito et al, 1990), Kluyveromyces bulgaricus (Hussain el al., 1986), Kluyveromyces marxianus (Bajpai and Margaritis, 1986), Pichia pastoris (Mbawala et al, 1990), Saccharomycodes ludwigii (M. Stratford, unpublished observation) and Schizosaccharomyces pombe (Johnson et al, 1988), flocculation genetics have only been studied, to date, in S. cerevisiae.…”

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confidence: 99%

Yeast flocculation: Reconciliation of physiological and genetic viewpoints

Stratford

1992

Yeast

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Yeast flocculation results from surface expression of specific proteins (lectins). Two flocculation phenotypes were suggested by physiological and biochemical tests, whereas genetic data suggested a larger number of mechanisms of flocculation. After reviewing the biochemistry, physiology and genetics of flocculation, a new hypothesis combining the data available from these different sources, is proposed. Flocculation results when lectins present on flocculent cell walls bind to sugar residues of neighbouring cell walls. These sugar receptors are intrinsic to the mannan comprising cell walls of Saccharomyces cerevisiae. Two lectin phenotypes were revealed by sugar inhibition studies. The gluco- and mannospecific NewFlo phenotype is not, as yet, found in genetically defined strains. Mannospecific flocculation (Flo1 phenotype) is found in strains containing the genes FLO1, FLO5 and FLO8. This phenotype is also found following mutation of the TUP1 or CYC8 loci, in previously non-flocculent strains. It is therefore proposed that the structural gene for mannospecific flocculation is common or possibly ubiquitous in non-flocculent strains and in consequence, FLO1, FLO5 and FLO8 are probably regulatory genes, exerting positive control over the structural gene. Flocculation expression requires lectin secretion to the cell surface. Many of the observed 'suppressions' of flocculation may be due to mutations of the secretory process, involved in transporting structural proteins to the cell wall. The possible involvement of killer L double-stranded RNA with flocculation is suggested, given the lectin properties of viral coat proteins and an association between L double-stranded RNA and the Flo1 phenotype.

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“…Flocculation often occurs upon nutrient limitation during late-exponential or stationary phase of growth and depends on pH, ethanol levels, or the carbon source available in the growth medium (63)(64)(65). Likewise, P. pastoris displayed flocculation in exponential and stationary growth phase (Fig.…”

Section: Discussionmentioning

confidence: 99%

A Novel Form of 6-Phosphofructokinase

Tanneberger

Kirchberger

Bär

et al. 2007

Journal of Biological Chemistry

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Classically, 6-phosphofructokinases are homo-and heterooligomeric enzymes consisting of ␣ subunits and ␣/␤ subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, ␣, ␤, and ␥. The sequence of the ␥ subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the ␥ subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the ␥ subunit is not required for enzymatic activity, the ␥ subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wildtype and ␥ subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the ␥ subunit. Therefore, we suggest that the ␥ subunit contributes to adaptation of P. pastoris to energy resources.The ATP-dependent 6-phosphofructokinase (EC 2.7.1.11, phosphofructokinase-1, ATP:D-fructose-6-phosphate 1-phosphotransferase (Pfk)) 2 catalyzes in many organisms the phosphorylation of fructose 6-phosphate (Fru 6-P) at position 1. The Pfk activity is generally being sensitive to a number of allosteric regulators, e.g. ATP, AMP, NH 4 ϩ , and fructose 2,6-bisphosphate (Fru 2,6-P 2 ). Therefore, this irreversible reaction is considered to be one of the rate-limiting steps of glycolysis (1-3). Most eukaryotic Pfks are heteromeric enzymes consisting of subunits, which evolved from a single ancestor gene by gene duplication and mutational events (4, 5). Specific amino acid residues involved in catalytic and regulatory functions of Pfk from Escherichia coli (6, 7) are conserved in yeast and mammalian Pfk genes. In eukaryotes the N-terminal half of a Pfk subunit obviously retained the catalytic function, whereas in the C-terminal half allosteric ligand binding sites have evolved from former catalytic and regulatory sites (4,8,9). This assumption is supported by studies with mutants of Saccharomyces cerevisiae expressing only the ␣ or the ␤ subunit of Pfk. It was demonstrated that one subunit type alone is able to form an enzymatically active Pfk entity in vivo (10, 11). Crystallographic analysis showed that an active bacterial Pfk consists of four identical subunits (12, 13). No high resolution structure of a eukaryotic Pfk is available yet. But electron microscopic studies with S. cerevisiae Pfk (ScPfk) at 10.8-Å resolution suggested an octameric enzyme assembly (14).Recently we co-purified a protein component together with the known Pfk ␣ and ␤ subunits (15-17) from the methylotrophic yeast Pichia pastoris. This unknown protein could only be separated under denaturating conditio...

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Acetolysis and 1H NMR studies on mannans isolated from very flocculent and weakly flocculent cells of Pichia pastoris IFP 206

Cited by 18 publications

References 16 publications

Yeast flocculation: Flocculation onset and receptor availability

Yeast flocculation: Flocculation onset and receptor availability

Yeast flocculation: Reconciliation of physiological and genetic viewpoints

A Novel Form of 6-Phosphofructokinase

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