FLO1, FLO5 and FLO11 Flocculation Gene Expression Impacts Saccharomyces cerevisiae Attachment to Penicillium chrysogenum in a Co-immobilization Technique

Abstract

A reoccurring flaw of most yeast immobilization systems that limits the potential of the technique is leakage of the cells from the matrix. Leakage may be due to weakly adherent cells, deterioration of the matrix, or to new growth and loss of non-adherent daughter cells. Yeast biocapsules are a spontaneous, cost effective system of immobilization whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum, creating hollow spheres that allow recovery and reutilization. This attachment is based on naturally occurring adherent properties of the yeast cell surface. We hypothesized that proteins associated with flocculation might play a role in adherence to fungal hyphae. To test this hypothesis, yeast strains with overexpressed and deleted flocculation genes (FLO1, FLO5, and FLO11) were evaluated for biocapsule formation to observe the impact of gene expression on biocapsule diameter, number, volume, dry mass, and percent immobilized versus non-immobilized cells. Overexpression of all three genes enhanced immobilization and resulted in larger diameter biocapsules. In particular, overexpression of FLO11 resulted in a five fold increase of absorbed cells versus the wild type isogenic strain. In addition, deletion of FLO1 and FLO11 significantly decreased the number of immobilized yeast cells compared to the wild type BY4742. These results confirm the role of natural adherent properties of yeast cells in attachment to fungal hyphae and offer the potential to create strongly adherent cells that will produce adherent progeny thereby reducing the potential for cell leakage from the matrix.

Keywords: FLO gene; Penicillium chrysogenum; Saccharomyces cerevisiae; co-immobilization; flocculation.

FIGURE 1

Yeast immobilization potential of null mutant strains versus BY4742 wt (blue) and overexpressed mutant strains versus FY23 wt (yellow). A and H after each strain name represent ADH2 and HSP30 promoters, respectively. (A) Yeast cells immobilized (dark color areas) and yeast cells non-immobilized (light color areas) considering each replica. Percentages of yeast cell immobilized out of total population are indicated for biocapsules made with each yeast strain. (B) Immobilized yeast cells per mg of biocapsule. (C) Immobilized yeast cells per biocapsule. In (A–C), homogenous groups are represented with alphabets where capital letters compare within biocapsules made with the null mutant and wild type, and lower-case letters compare biocapsules made with overexpressed mutants and wild type.

FIGURE 2

FY23 wt (left) and FY23 FLO11A (right) biocapsules (up) and non-immobilized yeast cells (down) after biocapsules were retrieved.

FIGURE 3

Multiple Variable Analysis (MVA) sunray plots and Principal Component Analysis (PCA) for biocapsules made with null mutants (A,B) and overexpressed mutants (C,D), respectively. In (A,C), each ray in the non-agon corresponds to one parameter: (1) free yeasts; (2) total immobilized yeasts; (3) % yeast immobilization; (4) immobilized yeast per biocapsule; (5) biocapsule number; (6) biocapsule total volume; (7) biocapsule diameter; (8) biocapsule dry weight; (9) immobilized yeast per mg biocapsule. The end of the ray is the mean value plus three standard deviations and the center the mean minus three standard deviations. (A,B) compare parameters between null strains and BY4742 wt; (C,D) compare parameters of overexpressed strains and FY23 wt. In (B,D), Principal Components Analyses Biplot were carried out with seven variables selected by their discrimination power among the yeast strains studied.

FIGURE 4

Average of biocapsule number of null mutant strains versus their wild type BY4742 wt (blue) and overexpressed mutant strains versus their wild type FY23 wt (yellow). Homogenous groups are represented with alphabets where capital letters compare within the null mutant and wild type, and lower-case letters compare within overexpressed mutants and wild type.

FIGURE 5

Biocapsule diameter measured in mm of null mutant strains versus their wild type BY4742 wt (blue) and overexpressed mutant strains versus their wild type FY23 wt (yellow). Homogenous groups are represented with alphabets where capital letters compare within the null mutant and wild type, and lower-case letters compare within overexpressed mutants and wild type.

FIGURE 6

Linear regression of biocapsule diameter and number. Null mutant strains and their wild type BY4742 wt are represented in blue and overexpressed mutant strains and their wild type FY23 wt in yellow.

FIGURE 7

Total volume of the biocapsules per replica of null mutants strains versus their wild type BY4742 wt (in blue) and overexpressed mutant strains versus their wild type FY23 wt (yellow). Homogenous groups are represented with alphabets where capital letters compare within the null mutant and wild type, and lower-case letters compare within overexpressed mutants and wild type.

FIGURE 8

Dry weight of the biocapsules of null mutants strains versus their wild type BY4742 wt (in blue) and overexpressed mutant strains versus their wild type FY23 wt (yellow). Homogenous groups are represented with alphabets where capital letters compare within the null mutant and wild type, and lower-case letters compare within overexpressed mutants and wild type.