1000/1000
Hot
Most Recent
The adaptive laboratory evolution (ALE) is a technique of strain optimization that assumes serial or continuous culturing of a particular yeast strain for many generations under selective pressure, such as high ethanol content, high osmolarity etc., thus directing the accumulation of mutants with desired phenotype. As compared to stochastic and laborious CSI techniques, ALE methods are more targeted and convenient . The power of this approach towards optimizing wine yeast is exemplified by generation of strains with altered production of important metabolites (ethanol, glycerol, succinic, and acetic acid) and more rapid sugar utilization, strains with increased sulfite tolerance and glycerol accumulation, strains with improved resistance towards KCL-induced osmotic stress with increased glycerol and reduced ethanol content, as well as enhanced viability and resveratrol production.
Modern industrial winemaking is based on the use of starter cultures of specialized wine strains of Saccharomyces cerevisiae yeast. Commercial wine strains have a number of advantages over natural isolates, and it is their use that guarantees the stability and reproducibility of industrial winemaking technologies. For the highly competitive wine market with new demands for improved wine quality, it has become increasingly critical to develop new wine strains and winemaking technologies. Novel opportunities for precise wine strain engineering based on detailed knowledge of the molecular nature of a particular trait or phenotype have recently emerged due to the rapid progress in genomic and “postgenomic” studies with wine yeast strains.
For laboratory strains of S. cerevisiae, an extensive and diverse set of tools for genetic engineering and directed modification of the genome has been developed quite a long time ago and are widely used for research in the fields of functional genomics, synthetic biology, biotechnology, and metabolic engineering [1]. At the same time, the application of such approaches for industrial strains faces a number of difficulties. These strains are usually polyploids and aneupoloids, poorly sporulate, there are no convenient auxotrophic markers for them, etc. [2].
The use of CRISPR-Cas genome editing systems can successfully overcome these limitations. The first work on the application of the CRISPR-Cas system for S. cerevisiae was published back in 2013 [3] and the advantages of this approach for yeast, in which the system of homologous recombination was already well developed, were at first not obvious. However, after overcoming a number of technical difficulties aimed at optimizing the expression and delivery of CRISPR-Cas system components, the system quickly gained popularity and is now successfully used in areas such as multiplex genome engineering, reprogramming transcription, creating synthetic genomes, etc. [4].
Examples of the successful application of CRISPR-Cas systems for industrial yeast strains relate to such aspects as the production of bioethanol from lignocellulosic raw materials, metabolic engineering for the production of vitamins and antibiotics, the improvement of aromatic and taste properties of beer, and a number of others [5][6][7]. From the point of view of food safety, it is fundamentally important that the use of CRISPR-Cas genome editing methods does not carry the risk of introducing foreign genes and genetic elements, markers of antibiotic resistance into the genomes of food yeast strains, i.e., the resulting strains are safe according to regulatory restrictions adopted in some countries.
One recent study describes the use of the CRISPR-Cas system for producing wine strains with reduced urea production. A group of scientists from Canada and Italy constructed derivatives of wine strains EC1118 and AWRI1796 defective in both alleles of the CAN1 gene [8][84]. The CAN1 gene encodes arginine permease, which along with GAP1 amino acids permease is responsible for the transport of arginine to yeast cells from the culture medium. During the subsequent stages of catabolism, arginine is cleaved by Car1p arginase to ornithine and urea, which is either excreted by Dur4p permease or converted to carbon dioxide and ammonia by Dur1p/Dur2p urea amidolyase. The resulting recombinant strains were characterized by reduced urea production (18–36% compared to the initial ones) under experimental micro-winemaking with the ability to ferment a synthetic substrate, although at a slightly reduced growth rate. The authors believe that further verification of the strains is necessary under the conditions of industrial winemaking. The advantage of introducing a mutation into the CAN1 gene compared to other methods of modifying arginine utilization pathways is that this technique is less sensitive to fluctuations in the content of nitrogen sources in the wort and less affects the growth parameters of yeast strains [7].
A promising area of application of genome editing methods is the directed change in the pathways of biosynthesis of aromatic compounds. Thus, in a recent work, yeast strains with increased production of phenylethyl acetate (PEA) were obtained using the CRISPR-Cas system [9]. PEA is an important aromatic compound that provides alcoholic drinks a pink and honey flavor. Genetic mapping methods first identified unique alleles of the FAS2 genes (encodes the α subunit of fatty acid synthase) and TOR1 (a growth regulator in response to the availability of a nitrogen source), linked to the trait of increased PEA production. Then, using CRISPR-Cas in commercial wine strains, wild alleles were replaced with mutant ones. As a result, the production of PEA increased by 70% [10].
In another work, the CRISPR-Cas system was used to reduce the production of 4-vinyl guaiacol (4VG) in a hybrid S. pastorianus/bayanus beer yeast strain [11]. It is known that 4VG is a sharp-tasting phenolic compound that spoils the organoleptic characteristics of beer. Formed from ferulic acid, 4VG is present in beer wort under the influence of yeast decarboxylase Fdc1p. Ale beer yeast strains do not produce 4VG due to the nonsense mutation in the FDC1 gene. Using the CRISPR-Cas system, the authors introduced a mutation characteristic of ale strains into all four copies of the FDC1 gene in the lager strain. The result was a strain containing a cis-gene mutation that lacks the ability to produce 4VG and has significant potential for use in the beer industry.
The CRISPR-Cas system is an extremely convenient tool for research in the field of functional genomics of wine strains. Until recently, the vast majority of experiments in the field of functional genomics of yeast were performed using laboratory strains. Nevertheless, according to the latest information from the SGD database (27 June 2020), when classified in terms of gene ontology, a significant number of yeast genes remain “unknown” (in the category “Biological Process”-1768 genes, 2548 genes in the category “Molecular Function” and 1298 genes in the cell compartment category). Such uncertainty is partly determined by the lack of specific conditions in which these genes are important. At the same time, these unknown genes experience regular changes in expression during many technological processes, including at different stages of wine fermentation (see, for example, [12]).
Characteristic changes in the expression pattern of a number of “unknown” genes were revealed in our recent work during the transcriptome analysis of the sherry strain at different stages of film formation [13]. CRISPR-Cas mediated genetic inactivation of “unknown” genes, allele replacement in wine strains of yeast can significantly clarify their role in various winemaking processes, and will help to create strains with improved characteristics(Table 1).
Table 1. Selected metabolically-engineered yeast strains and their oenology-related phenotypes.
Strain |
Genetic Modification |
Oenology-Related Trait |
Ref. |
ML01 |
Overexpression of S.pombe mae1 gene O.oeni mleA gene |
Malolactic fermentation |
[14] |
ECMo01 |
Overexpression of S.cerevisiae DUR1,2 gene |
Reduced ethyl carbamate content |
[15] |
AWRI 1631 |
Deletion of MFA2 gene |
Improved fermentation efficiency under nitrogen limitation |
[16] |
C911D |
Deletion of ECM33 gene |
Improved fermentation efficiency under nitrogen limitation |
[17] |
S288C |
Overexpression of S.cerevisiae YOL155c and YDR055w genes |
reduced haziness during fermentation |
[18] |
EC1118 |
Deletion of KNR4 gene |
reduced haziness during fermentation, retaining good fermentation performance |
[19] |
VIN13 |
Overexpression of Butyrivibrio fibrisolvens end1 gene, Aspergillus niger xynC gene |
decrease in wine turbidity, increase in colour intensity, increase in phenolic compounds |
[20] |
VIN13 |
Overexpression of Erwinia chrysanthemi pelE gene, Erwinia carotovora peh1 gene |
decrease in phenolic compounds |
[20] |
ICV16, ICV27 |
Overexpression of S. cerevisiae HSP26 and YHR087W genes |
Improved Stress resistance and fermentation efficiency |
[21] |
PYCC 5484 |
Overexpression of 925–963 segments of TDH1 and TDH2/3 ORFs |
Secretion of AMPs, inhibiting D.bruxellensis growth |
[22] |
Sigma1278 |
Overexression of A. niger GOX gene |
Reduction of sugar content in juice |
[23] |
V5.TM6*P. |
Overexpression of chimeric HXT1-HXT7 gene in a hxt null strain |
decreased ethanol production, increased biomass under high glucose conditions |
[24] |
MC42 |
Deletion of ADH1, ADH3,ADH4 genes, ADSH2 gene mutations |
66% reduction of ethanol yield, increased glycerol production |
[25] |
CEN.PK 113-7D |
Deletion of TPI1 gene |
Unable to grow on glucose, growth on mixed substrates |
[26] |
YSH l.l.-6B |
Deletion of PDC2 gene, overexpression of GPD1 gene |
Reduction of glucose catabolism, 6-7-fold increase in glycerol formation |
[27] |
AWRI1631 |
GPD1 overexpression, ALD6 deletion * |
Decreased ethanol production |
[28] |
BY4742, VIN13 |
Screening of EOROSCARF deletion collection, weak TPS overexpression |
10% reduction in ethanol yield, increased glycerol, trehalose production |
[29] |
CMBS33, BY4742 |
Analysis of ATF1,2 knockouts in the lab strain, constitutive ATF1,2 overexpression in lager strains |
Reduction in acetate esters production in ATF1,2 deletion strains, enhanced production of volatile esters in overexpression strains |
[30] |
T73-4 |
Overexpression of Ocimum basilicum (sweet basil) geraniol synthase (GES) gene |
Increased geraniol production during fermentation, 230-fold increased total monoterpene content |
[31] |
VIN13 |
Overexpression of A. awamori arabinofuranosidase, A. kawachii β-glucosidase. |
increased release of citronellol, linalool, nerol and α-terpineol. |
[32] |
WY1 |
Overexpression of BDH1,2 genes |
Decreased diacetyl, increased acetoin, butanediol contents |
[33] |
AWRI |
Overexpression of RtPAL, AtC4H, At4CL, RtBAS genes for frambion biosynthesis |
Frambion production at 0.68 mg/L simultaneously with chardonnay wine fermentation |
[34] |
CEN.PK 113-7D |
Overexpression of AtPAL2, AtC4H, At4CL, VvVST1 gene for resveratrol biosynthesis, complex strain and cultivation optimization strategy |
Yeast-based de novo resveratrol production from glucose at 800 mg/l level |
[35] |
133d |
Overexpression of FLO11 gene using different promoter variamts |
Improved velum formation |
[36] |
P3-D5 |
Deletion of CCW14, YGP1 genes in a flor strain |
Impaired velum formation |
[37] |
FJF206, FJF414, B16 |
Overexpression of SOD1, SOD2, HSP12 in flor strains |
increased superoxide dismutase, catalase, gluthathione peroxidase activities, increased oxidative stress resistance, quicker velum formation,slight decrease in ethanol and increase in acetaldehyde content |
[38] |
EC1118, AWRI1796 |
Crispr-cas9 mediated inactivation of CAN1 gene |
Reduced ethyl-carbamate formation |
[9] |
BTC.1D |
Crispr-cas9 mediated allele exchange for FAS2 and TOR1 genes in wine strain |
Increased phenyl-ethyl acetate formation |
[10] |
W34/70 |
Crispr-cas9 mediated allele exchange for FDC1 gene in lager strain |
Decreased 4-vinyl guaiacol formation |
[11] |
* other modifications had non-significant effects.