Isolation and Identification of Non-Saccharomyces Yeast Producing 2-Phenylethanol and Study of the Ehrlich Pathway and Shikimate Pathway
<p>Volatile compounds in stationary phase of fermentation produced by strain R5.</p> "> Figure 2
<p>Colony morphology (<b>A</b>) and cell morphology (<b>B</b>) of strain R5.</p> "> Figure 3
<p>Phylogenetic trees based on of 26S rRNA sequences (<b>A</b>) and ITS gene sequences (<b>B</b>).</p> "> Figure 4
<p>KOG function categories of strain R5. Note: the genome protein sequences of strain R5 were compared with the KOG database by Blastp software, and its functions annotated by protein sequences were sorted.</p> "> Figure 5
<p>Prediction for 2-PE synthesis pathway of strain R5. Note: PEP: phosphoenolpyruvate, E4P: erythrose-4-phosphate, DAHP: 3-deoxy-d-arabinoheptanose-7-phosphate, DHQ: 3-dehydroquinic acid, DHS: 3-dehydroshikimic acid, SHK: shikimate, S3P: shikimate-3-phosphate, EPSP: 5-enolpyruvate 3-phosphate, CHR: chorismite, PPA: phenylpyruvate, PAAL: phenylacetaldehyde, 2-PE: 2-phenyl alcohol, L-phe: L-phenylalanine.</p> "> Figure 6
<p>Effect of different nitrogen sources on the growth and 2-PE synthesis of strain R5.</p> "> Figure 7
<p>mRNA expression levels of key genes of strain R5 in different nitrogen sources. Note: * represents <span class="html-italic">p</span> < 0.05; ** represents <span class="html-italic">p</span> < 0.01, and ns represents no significance.</p> "> Figure 8
<p>Effect of exogenous 2-PE on strain R5 growth.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Isolation of Yeast Strains
2.2. Screening of Yeast Strains Producing 2-PE
2.3. Identification of Yeast Strains Producing 2-PE
2.3.1. Observation of Morphology
2.3.2. Physiological Characteristics
2.3.3. Molecular Biological Identification
2.4. Whole-Genome Sequencing, Assembly, and Annotation of Strain R5
2.4.1. Genomic DNA Extraction and Sequencing
2.4.2. Whole-Genome Data Processing and Analysis
2.5. Study of the Synthesis Pathway of 2-PE
2.6. Quantitative Real-Time PCR (qRT-PCR)
2.7. Tolerance of Yeast Strains to 2-PE
3. Results and Discussion
3.1. Isolation and Screening of Yeast Strains Producing 2-PE
3.2. Identification of Morphology, Physiology, and Molecular Biology
3.3. Whole-Genome Sequencing, Assembly, and Annotation of Strain R5
3.4. Identification Synthesis Pathway of 2-PE by Strain R5
3.5. Identification of Potential Genes Involved in 2-PE Synthesis by qRT-PCR
3.6. Effect of 2-PE Stress on the Growth of R5 Strain
4. Conclusions
- (1)
- In this study, a yeast strain was isolated and screened from pear peels that was capable of producing 2-PE. Based on morphology observation, analysis of physiological characteristics, and molecular biological identification, strain R5 was identified as S. bacillaris.
- (2)
- Subsequently, through the analysis of whole-genome sequencing and comparison with the KEGG database, we found that strain R5 possesses a metabolic pathway producing 2-PE. To further investigate the synthesis pathway of 2-PE, strain R5 was inoculated into three M3 media. When strain R5 was cultured in M3 (Phe) medium, 2-PE was biosynthesized from L-phe via the Ehrlich pathway. When strain R5 was inoculated in M3 (NH4+) medium, 2-PE was produced from glucose through the shikimate pathway. In M3 (Phe) medium, the maximum concentration of 2-PE reached 1.28 g/L, which was 16-fold and 2.29-fold higher than that in M3 (NH4+) and M3 (Phe + NH4+) media, respectively. These results show that the Ehrlich pathway is the main synthetic pathway for producing 2-PE in strain R5.
- (3)
- The qRT-PCR results revealed that the transport of L-phe was inhibited when both NH4+ and Phe were present in the medium. The key gene catalyzing the dehydrogenation of benzaldehyde into 2-PE is ADH5. And genes ADH5, PDC, hisC, and GOT1 are not sensitive to nitrogen metabolism inhibition. These findings provide evidence that strain R5 is capable of biotransforming L-phe into 2-PE.
- (4)
- In summary, all the above results illustrated that the Ehrlich pathway and shikimate pathway synthesize 2-PE in strain R5, mainly for the Ehrlich pathway, which had not been previously investigated. These findings fill the research gap relating to the synthesis pathway of 2-PE in strain R5 and provide a solid basis for future research on the application of non-Saccharomyces yeasts in the wine industry.
5. Accession Number
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Glucose (g/L) | Sucrose (g/L) | YNB (g/L) | L-phe (g/L) | (NH4)2SO4 (g/L) | MgSO4·7H2O (g/L) | |
---|---|---|---|---|---|---|
M3 (Phe) | 30 | 8 | 1.7 | 9 | - | 0.5 |
M3 (Phe + NH4+) | 30 | 8 | 1.7 | 4.5 | 2.25 | 0.5 |
M3 (NH4+) | 30 | 8 | 1.7 | - | 4.5 | 0.5 |
Primers | Sequences |
---|---|
ENO F | 5′-TGCTATTGACGCTGCTGGTTA-3′ |
ENO R | 5′-GCCTTGGACTTGTCGGAGTTA-3′ |
YAT F | 5′-GGCGAGGTAGCAGTTAGATTC-3′ |
YAT R | 5′-TGCGGCGGTCAATTCCAA-3′ |
GOT1 F | 5′-TCACCAGTCAACTCGCTTACC-3′ |
GOT1 R | 5′-CAATTTACCACGCAGGGCTTT-3′ |
hisC F | 5′-GCACCTTCAATGGCACCACTA-3′ |
hisC R | 5′-GTCAGCAGTAAGAACGGAGA-3′ |
PDC F | 5′-GGTGTCGTTGATGAGGTTGAGA-3′ |
PDC R | 5′-GGAAGCGTAGAAGGCGTGAA-3′ |
ADH5 F | 5′-TGGCTCAACCTATCAAGTGTCA-3′ |
ADH5 R | 5′-ACCAAGAACAGAAGGCGTGAA-3′ |
Items | Results | Items | Results |
---|---|---|---|
D-pine trisaccharides assimilate | − | L-lysine arylamase | − |
L-malic acid assimilate | + | D-sorbitol assimilate | + |
2-Keto-gluconate assimilate | − | L-rhamnose assimilate | − |
Glucuronic acid assimilate | + | Xylitol | − |
Erythritol assimilate | − | D-sorbitol assimilate | + |
Glycerol assimilate | − | Sucrose assimilate | + |
D-brown sugar assimilate | − | Urease | − |
β-N-acetyl glucosaminidase | + | α-glucosidase | − |
Myric acid assimilate | + | Tyrosine arylamase | − |
Amygdalin assimilate | − | D-trehalose assimilate | + |
α-galactose assimilate | + | Nitrate assimilate | − |
Gentiobiose assimilate | + | L-arabinose assimilate | + |
D-glucose assimilate | + | Lactose assimilate | + |
D-galacturaldehyde assimilate | + | Aesculin hydrolysis | + |
Methyl glucoside assimilate | − | L-Glutamate assimilate | + |
D-cellobiose assimilate | + | Xylose assimilate | − |
γ-glutamyl transferase | − | DL-lactate assimilate | − |
D-maltose assimilate | + | Acetate assimilate | − |
D-raffinose assimilate | − | Citrate assimilate | − |
PNP-n-acetyl-BD-galactosidase | − | Arginine | + |
D-mannose assimilate | + | L-proline assimilate | − |
D-melibiose assimilate | − | Leucine arylamase | − |
N-Acetyl-Glucosamine assimilate | − | D-gluconate assimilate | − |
Assembly Feature | R5 |
---|---|
Assembled sequence (bp) | 9,474,513 |
No. of scaffolds | 250 |
Sequence depth | 204.03 |
Maximum contig length (bp) | 4,175,501 |
N50 length (bp) | 3,806,193 |
N90 length (bp) | 1,100,691 |
GC content in genome (%) | 39.62 |
Strain | S. bacillaris R5 | S. bacillaris PAS13 | S. cerevisiae S288c |
---|---|---|---|
Ploidy | n | N | n |
Genome size (Mb) | 9.47 | 9.4 | 12.3 |
GC content in genome (%) | 39.62 | 39.45 | 38.3 |
Total number of CDS | 3991 | 4321 | 5769 |
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Zhou, R.; Song, Q.; Xia, H.; Song, N.; Yang, Q.; Zhang, X.; Yao, L.; Yang, S.; Dai, J.; Chen, X. Isolation and Identification of Non-Saccharomyces Yeast Producing 2-Phenylethanol and Study of the Ehrlich Pathway and Shikimate Pathway. J. Fungi 2023, 9, 878. https://doi.org/10.3390/jof9090878
Zhou R, Song Q, Xia H, Song N, Yang Q, Zhang X, Yao L, Yang S, Dai J, Chen X. Isolation and Identification of Non-Saccharomyces Yeast Producing 2-Phenylethanol and Study of the Ehrlich Pathway and Shikimate Pathway. Journal of Fungi. 2023; 9(9):878. https://doi.org/10.3390/jof9090878
Chicago/Turabian StyleZhou, Rong, Qingyi Song, Huili Xia, Na Song, Qiao Yang, Xiaoling Zhang, Lan Yao, Shihui Yang, Jun Dai, and Xiong Chen. 2023. "Isolation and Identification of Non-Saccharomyces Yeast Producing 2-Phenylethanol and Study of the Ehrlich Pathway and Shikimate Pathway" Journal of Fungi 9, no. 9: 878. https://doi.org/10.3390/jof9090878