Gene expression and biochemical analysis of cheese-ripening yeasts: focus on catabolism of L-methionine, lactate, and lactose

Abstract

DNA microarrays of 86 genes from the yeasts Debaryomyces hansenii, Kluyveromyces marxianus, and Yarrowia lipolytica were developed to determine which genes were expressed in a medium mimicking a cheese-ripening environment. These genes were selected for potential involvement in lactose/lactate catabolism and the biosynthesis of sulfur-flavored compounds. Hybridization conditions to follow specifically the expression of homologous genes belonging to different species were set up. The microarray was first validated on pure cultures of each yeast; no interspecies cross-hybridization was observed. Expression patterns of targeted genes were studied in pure cultures of each yeast, as well as in coculture, and compared to biochemical data. As expected, a high expression of the LAC genes of K. marxianus was observed. This is a yeast that efficiently degrades lactose. Several lactate dehydrogenase-encoding genes were also expressed essentially in D. hansenii and K. marxianus, which are two efficient deacidifying yeasts in cheese ripening. A set of genes possibly involved in l-methionine catabolism was also used on the array. Y. lipolytica, which efficiently assimilates l-methionine, also exhibited a high expression of the Saccharomyces cerevisiae orthologs BAT2 and ARO8, which are involved in the l-methionine degradation pathway. Our data provide the first evidence that the use of a multispecies microarray could be a powerful tool to investigate targeted metabolism and possible metabolic interactions between species within microbial cocultures.

FIG. 1.

pH and growth of three yeasts cultivated separately (open symbols) and in coculture (closed symbols) over time. (a) □, K. marxianus alone; (b) ○, D. hansenii alone; (c) ▵, Y. lipolytica alone; (d) ▪, K. marxianus; •, D. hansenii; and ▴, Y. lipolytica in coculture. pH levels. Error bars represent standard deviations calculated on the average values of six determinations.

FIG. 2.

Distribution of spot intensities (arbitrary units) under different experimental conditions: (a) Cy3-cDNA prepared from D. hansenii mRNA; (b) Cy3-cDNA prepared from K. marxianus mRNA; (c) Cy3-cDNA prepared from Y. lipolytica mRNA. The genes were ranked according to the intensities of the corresponding hybridization signals. The values were then arranged into four sets: Class <250; 250 to 2,500; 2,500 to 25,000; and 25,000 to 70,000, with increasing hybridization values. Black histograms, D. hansenii genes. Hatched histograms, K. marxianus genes. White histograms, Y. lipolytica genes.

FIG. 3.

Scatter plots of the signal intensities obtained from mRNA of D. hansenii in pure culture (pure) versus mRNA of D. hansenii in artificial mixed culture (pooled) with K. marxianus and Y. lipolytica.