Submitted:
04 June 2025
Posted:
04 June 2025
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Abstract
Keywords:
1. Introduction
2. GMO-Free Genetic Improvement Techniques
2.1. Classical Genetic Improvement Techniques for Non-GMO Yeasts
2.1.1. Random Mutagenesis
2.1.2. Sexual Hybridization
2.2. Innovative Genetic Improvement Techniques in Fermentation
2.2.1. Adaptive Laboratory Evolution - ALE
2.2.2. Big Data, AI and Omics
2.2.3. Synthetic Microbial Communities
2.3. GMO-Based Genetic Improvement Techniques
2.3.1. Synthetic Biology and CRISPR/Cas9
2.3.2. Ethical and Commercial Challenges
3. Conclusion
Acknowledgments
References
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| Technique | Pros | Cons |
|---|---|---|
|
CRISPR-Cas9 GMO |
Precise and efficient genome editing; Allows for modifications of multiple genes at once; highly versatile across different yeast strains. |
Ethical concerns around gene editing; Requires optimization for different yeast species; Requires advanced understanding of yeast genetics |
|
Synthetic Microbial Communities (SMC) GMO or Non-GMO (depending on strains used) |
Enables creation of yeast strains consortia with complementary traits that work together; Can improve metabolic networks. |
Requires compatibility between strains, including nutrient requirements; If OGM strains are used, there are ethical concerns around genetic modifications. |
|
Hybridization Non-GMO |
Simple method to combine beneficial traits from different yeast strains; Heterosis compared with parents; Well-established and cost-effective. |
May result in sterility or instability of the hybrid offspring; Difficult to obtain for poorly sporulating strains; undesirable characteristics may emerge. |
|
Mutagenesis Non-GMO |
Generates a wide variety of potential phenotypes; Relatively inexpensive and straightforward; Suitable for monogenic phenotypes. |
Random outcomes make it difficult to predict results; Can introduce harmful mutations or undesired traits; Not suitable for polygenic or complex phenotypes. |
|
Adaptive Laboratory Evolution (ALE) Non-GMO |
Mimics natural selection, leading to improved fitness-related traits over time; Suitable for phenotypes that can be directly selected under controlled environments. |
Time-consuming and labor-intensive; Unintended side effects may occur, as it is difficult to control specific outcomes; May not be suitable for traits without clear selection markers. |
|
Multi-Omics and AI Integration Non-GMO |
Provides a comprehensive view of yeast metabolism and gene expression; Unintended side effects may occur, as it is difficult to control specific outcomes. |
Requires large data sets and significant computational resources; Interpreting the data can be complex and requires expert knowledge. |
| Objective | Work |
|---|---|
| Increased ethanol tolerance | [31] |
| Elimination of phenolic off-flavors (POF) | [32] |
| Enhanced nitrogen source utilization | [33] |
| Increased mannoprotein release | [34] |
| Reduced volatile acidity | [35] |
| Increased aroma compound concentration (esters) | [36] |
| Reduction of isoamyl alcohol (3-methylbutanol) production | [37] |
| Objective | Work |
|---|---|
| Improved fermentation and aroma production in lager hybrids | [19,30,39,40] |
| Elimination of undesirable traits (e.g., SO₂ formation, excessive foam production) | [41] |
| Enhanced wine quality and fermentation rates via interspecific hybridization | [23,42] |
| Objective | Work |
|---|---|
| Increased tolerance to acetic acid | [53] |
| Selection of Atf2-overexpressing strains for ester formation and pregnenolone detoxification | [50] |
| Improved beer yeast performance via UV mutagenesis and high-gravity wort fermentations | [54] |
| Enhanced ethanol tolerance in hybrid yeasts | [55] |
| Genomic adaptations linked to chromosomal duplications and mutations in IRA2 and UTH1 | [56,57] |
| Increased sulfite tolerance in T. delbrueckii | [58] |
| Enhanced sulfite resistance in B. bruxellensis | [59] |
| Adaptation of Saccharomyces variants to overcome premature yeast flocculation (PYF) | [60] |
| Reduction of phenolic off-flavors via PAD1 and FDC1 mutations | [44,45] |
| Increased glycerol production for lower ethanol wines | [61] |
| Development of yeast strains producing higher levels of glutathione (GSH) | [62] |
| Enhanced yeast flocculation for easier removal after fermentation | [63] |
| Application | Involved Species | Work |
|---|---|---|
| Alcoholic fermentation in beer production | Saccharomyces cerevisiae | [86] |
| Bio-acidification and microbial control in beer | Lactic acid bacteria (LAB) | [87] |
| Malolactic fermentation for flavor complexity in wine | Oenococcus oeni | [86] |
| Adaptation to vineyard microbial terroir | Various LAB and yeast species | [88] |
| Yeast community shifts during grape must fermentation | K. apiculate, C. stellata, C. pulcherrima | [89] |
| Influence of nutrient scarcity, oxygen availability, and ethanol on fermentation | Saccharomyces cerevisiae and other yeasts | [90,91] |
| Ecological interactions driving fermentation outcomes | Multiple yeast species | [92] |
| Yeast-yeast interactions, including S. cerevisiae with non-Saccharomyces species | Wickerhamomyces anomalus, Hanseniaspora vineae | [93] |
| Persistence of certain non-Saccharomyces yeasts in vineyard ecosystems | Starmerella bacillaris, Lachancea thermotolerans | [94] |
| Yeast ecosystem modulation by S. cerevisiae | Various non-Saccharomyces species | [80] |
| Optimization of yeast interactions for improved fermentation | Selected co-cultures of Saccharomyces and non-Saccharomyces | [89] |
| Process | Technique | References |
|---|---|---|
| GPD1 overexpression and ALD6 delation to reduce reduce alcohol yield in wine yeast | Episomal vector; KanMX deletion cassette | [94] |
| Expression of extracellular hydrolytic enzymes to improve juice extraction and release primary aromas | Episomal vector constructed by restriction cloning | [106] |
| Reduction of urea and ethyl carbamate formation | CRISPR/Cas9 | [107] |
| Overexpression of LEU4 (α-isopropylmalate synthase) in sake yeast to increase isoamyl alcohol and esters | Plasmid multicopy integration at chromosome level | [108] |
| Reduction of ethyl carbamate production | Plasmid distruption | [109] |
| Heterologous expression of pediocin to increase resistance to wild yeasts and bacteria | Episomal vector constructed by restriction cloning | [110] |
| Expression of acetolactate decarboxylase (ALDC) to reduce diacetyl formation | Episomal vector constructed by restriction cloning | [111] |
| Expression of malolactic enzymes to degrade malate and integrate malolactic fermentation | Episomal vector constructed by restriction cloning | [112] |
| Engineering yeast strains to produce hop monoterpenes | Plasmids obtained by Golden Gate assembly | [113] |
| Engineering yeast strains to produce methyl anthranilate with grape aroma | CRISPR/Cas9 | [114] |
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