Clarification of inhibitory mechanism of proline utilization infermentations and its application
Akira NISHIMURA
Graduate School of Science and Technology, Nara Institute of Science andTechnology
Research objective
Yeasts play an important role in ethanol production and dictating the aroma and taste ofalcoholic beverages. Thus, the quality of alcoholic beverages strongly depends on themetabolic characteristics of yeast cells. In the wine-making process, grapes must contain asufficient quantity of sugar for the maximal growth of wine yeasts. In contrast, the total
nitrogen content of the grapes must range from 60 to 2,400 mg/L, which limits yeast growth.
Therefore, nitrogen availability is an essential parameter of wine fermentation.
Proline is the predominant amino acid in grapes and is considered a potentiallysignificant and effective source of nitrogen. However, the wine yeast Saccharomyces
cerevisiae cannot utilize proline during wine fermentation1). This often leads to nitrogen
deficiency during fermentation and proline accumulation in wine. To prevent nitrogen
deficiency, ammonium salts, such as diammonium phosphate, are frequently supplied during
the fermentation processes. Such additives can impact yeast metabolism and thus, influence
the quality of the wine by altering the aroma and taste proﬁles. Moreover, supplementation
with ammonium salt causes the accumulation of ethyl carbamate, a carcinogen. In addition,
excess proline in wine has negative effects on wine quality, because residual proline leads toa decrease in the sweetness and acidity of wine. Hence, a yeast strain that can utilize proline
is a promising candidate for overcoming nutrient-related fermentation problems, thereby
improving wine fermentation ability and wine quality.
Methods
1. Strains
The wine yeast wild-type strain (provided by Dr. Walker, University of Adelaide), a proline
auxotrophic strain (pro3Δ/pro3Δ), and a cdc25Glu916* mutant strain (cdc25Glu916Stop/cdc25Glu916Stop) were used in this study.
2. Measurement of residual amino acids
The yeast strains were inoculated into white grape (WG) medium starting from an opticaldensity at 600 nm (OD600) of 1.5. After incubation under static conditions at 30°C for thedurations indicated in the figure legends, the supernatants were collected by centrifugation.
The residual amino acid content of each supernatant was determined using an amino acidanalyzer (JLC-500/V; JEOL, Tokyo, Japan).
3. Isolation of proline-utilizing mutants under WG medium
Strain pro3Δ/pro3Δ was plated on WG medium. After 3 days at 30°C, the two colonies werepicked, and named P1 and P2. For whole-genome sequencing, genomic DNA was extracted
from cells using a Dr. GenTLE (from yeast) High Recovery Kit (Takara Bio, Kusatsu, Japan).
Libraries for sequencing analysis were prepared using an NEB Next Ultra DNA Library PrepKit (New England Biolabs, Ipswich, MA, USA), and paired-end reads of 150 bp wereproduced using a NovaSeq 6000 (Illumina, San Diego, CA, USA). Sequencing was performed
by a commercial DNA sequencing service (Rhelixa, Tokyo, Japan).
4. cAMP determination
Each strain was suspended in WG medium and the cells were collected by centrifugation at
the time points indicated in the figure legends. Metabolites were extracted from the cells
adding trichloroacetic acid (final concentration 5%). cAMP levels in the extracts weredetermined using a cAMP EIA kit (Cayman Chemical, Ann Arbor, MI, USA) according to themanufacturer’s instructions. Values were normalized to OD600 units.
5.

Detection of proteins phosphorylated by protein kinase A

Protein kinase A (PKA)-phosphorylated proteins were identified by western blotting using
an anti-phosphorylated PKA consensus RRxS/T rabbit antibody (Cell Signaling Technology,Danvers, MA, USA).
Results
We performed genetic screening to identify genes involved in the inhibition of proline
utilization. By screening with pro3Δ/pro3Δ and WG medium, we obtained two spontaneous
mutants (which we named P1 and P2) that were able to grow on WG medium. The growthphenotypes of P1 and P2 were confirmed in liquid WG medium. Importantly, P1 and P2 clearly
consumed proline under static conditions in WG medium, whereas the WT/WT andpro3Δ/pro3Δ strains did not consume proline. Whole-genome sequencing analysis revealed
that both P1 and P2 had a T nucleotide at position 2746, only at the CDC25 locus encoding aguanine nucleotide exchange factor (GEF) of Ras1/2, which is involved in the glucose
response. In contrast, pro3Δ/pro3Δ and laboratory yeast strains with publicly available
genomic information (Saccharomyces Genome Database: https://www.yeastgenome.org/)
have a G nucleotide at the same position. This G to T mutation (2746 G>T) led to theoccurrence of a stop codon at position 916 (Glu916*), indicating that P1 and P2 have ahomozygous nonsense mutation in CDC25. cdc25Glu916* encodes a Cdc25 variant with a GEF
domain deletion, suggesting that Cdc25 Glu916* is a loss-of-function mutant. Next, we
examined the effects of the cdc25Glu916* mutation on Ras/PKA signaling in WG medium.
cAMP levels in the WT/WT strain transiently increased for up to 10 min upon transfer to WG
medium, but cAMP levels in the cdc25Glu916Stop/ cdc25Glu916Stop strain did not. The
phosphorylation levels of PKA substrates in the WT/WT strain increased upon transfer to WG
medium. The cdc25Glu916* mutation resulted in no significant change in the phosphorylation

levels of PKA substrates upon transfer to WG medium. Finally, we determined proline
consumption by the WT/WT and cdc25Glu916Stop/ cdc25Glu916Stop strains. The WT/WT strain did
not consume proline in WG medium. In contrast, the cdc25Glu916Stop/ cdc25Glu916Stop strains
showed clear consumption of proline. Activation of the Ras/PKA pathway inhibits proline
utilization in wine yeast (Figure 1)2). Herein, we propose that crosstalk occurs between carbon
metabolism and proline utilization.

Figure 1 Regulation of proline utilization by the PKA pathway.
Activation of the Ras/PKA pathway via Cdc25 by glucose leads to the inhibition ofproline utilization.
Conclusion
Wine is a traditional fermented beverage brewed with wine yeast (most commonly S.
cerevisiae). Product innovation is a vital source of competitive advantage, and wine is no
exception to this principle. Therefore, winemakers continue to pursue new wine yeast strains
to meet the demand for high-quality wines. Proline is the predominant amino acid in grapemust, but is poorly utilized by yeast during the wine-making processes. This phenomenon
negatively affects winemaking quality and productivity. Yeast cells have been known for morethan 50 years to consume little proline during wine fermentation; however, the detailed
underlying mechanisms remain unclear. Our data suggest that the carbon sources present
during wine fermentation activate PKA signaling via the Cdc25/Ras pathway, which in turn,leads to the inhibition of proline utilization by yeast. In short, they suggested the existence ofcrosstalk between carbon and proline metabolism. Our results hold promise for thedevelopment of wine yeast strains that are capable of efficiently assimilating proline during
fermentation.
References
1) Valero, E., Millán, C., Ortega, J. M., and Mauricio, J. C. (2003) Concentration ofamino acids in wine after the end of fermentation by Saccharomyces cerevisiae strains.
J. Sci. Food Agric. 83: 830-835.

2) Nishimura, A., Ichikawa, K., Nakazawa, H., Tanahashi, R., Morita, F., Sitepu, I.,
Boundy-Mills, K., Fox, G., and Takagi, H. (2022) The Cdc25/Ras/cAMP-dependent
protein kinase A signaling pathway regulates proline utilization in wine yeast
Saccharomyces cerevisiae under a wine fermentation model. Biosci. Biotechnol.
Biochem. 86: 1318-1326.