Structure-guided engineered urethanase from Candida parapsilosis with pH and ethanol tolerance to efficiently degrade ethyl carbamate in Chinese rice wine.

Urethane hydrolase can degrade the carcinogen ethyl carbamate (EC) in fermented food, but its stability and activity limit its application. In this study, a mutant G246A and a double mutant N194V/G246A with improved cpUH activity and stability of Candida parapsilosis were obtained by site-directed mutagenesis. The catalytic efficiency (Kcat/Km) of mutant G246A and double mutant N194V/G246A are 1.95 times and 1.88 times higher than that of WT, respectively. In addition, compared with WT, the thermal stability and pH stability of mutant G246A and double mutant N194V/G246A were enhanced. The ability of mutant G246A and double mutant N194V/G246A to degrade EC in rice wine was also stronger than that of WT. The mutation increased the stability of the enzyme, as evidenced by decreased root mean square deviation (RMSD) and increased hydrogen bonds between the enzyme and substrate by molecular dynamics simulation and molecular docking analysis. The molecule modification of new cpUH promotes the industrial process of EC degradation.

Reduced production of Ethyl Carbamate in wine by regulating the accumulation of arginine in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a multisite carcinogenic compound, is naturally produced from urea and ethanol in alcoholic beverages. In order to reduce the content of EC in wine, the accumulation of arginine in Saccharomyces cerevisiae was regulated by genetic modifying genes involved in arginine transport and synthesis pathways to reduce the production of urea. Knockout of genes encoding arginine permease (Can1p) and amino acid permease (Gap1p) on the cell membrane as well as argininosuccinate synthase (Arg1) respectively resulted in a maximum reduction of 66.88% (9.40 µg/L) in EC, while overexpressing the gene encoding amino acid transporter (Vba2) reduced EC by 52.94% (24.13 µg/L). Simultaneously overexpressing Vba2 and deleting Arg1 showed the lowest EC production with a decrease of 68% (7.72 µg/L). The yield of total higher alcohols of the mutants all decreased compared with that of the original strain. Comprehensive consideration of flavor compound contents and sensory evaluation results indicated that mutant YG21 obtained by deleting two allele coding Gap1p performed best in must fermentation of Cabernet Sauvignon with the EC content low to 9.40 µg/L and the contents of total higher alcohols and esters of 245.61 mg/L and 41.71 mg/L respectively. This study has provided an effective strategy for reducing the EC in wine.

Formation and hazard of ethyl carbamate and construction of genetically engineered Saccharomyces cerevisiae strains in Huangjiu (Chinese grain wine).

Huangjiu, a well-known conventional fermented Chinese grain wine, is widely consumed in Asia for its distinct flavor. Trace amounts of ethyl carbamate (EC) may be generated during the fermentation or storage process. The International Agency for Research on Cancer elevated EC to a Class 2A carcinogen, so it is necessary to regulate EC content in Huangjiu. The risk of intake of dietary EC is mainly assessed through the margin of exposure (MOE) recommended by the European Food Safety Authority, with a smaller MOE indicating a higher risk. Interventions are necessary to reduce EC formation. As urea, one of the main precursors of EC formation in Huangjiu, is primarily produced by Saccharomyces cerevisiae through the catabolism of arginine, the construction of dominant engineered fermentation strains is a favorable trend for the future production and application of Huangjiu. This review summarized the formation and carcinogenic mechanism of EC from the perspectives of precursor substances, metabolic pathways after ingestion, and risk assessment. The methods of constructing dominant S. cerevisiae strains in Huangjiu by genetic engineering technology were reviewed, which provided an important theoretical basis for reducing EC content and strengthening practical control of Huangjiu safety, and the future research direction was prospected.

Comparative analysis of hippocampal extracellular space uncovers widely altered peptidome upon epileptic seizure in urethane-anaesthetized rats.

BACKGROUND: The brain extracellular fluid (ECF), composed of secreted neurotransmitters, metabolites, peptides, and proteins, may reflect brain processes. Analysis of brain ECF may provide new potential markers for synaptic activity or brain damage and reveal additional information on pathological alterations. Epileptic seizure induction is an acute and harsh intervention in brain functions, and it can activate extra- and intracellular proteases, which implies an altered brain secretome. Thus, we applied a 4-aminopyridine (4-AP) epilepsy model to study the hippocampal ECF peptidome alterations upon treatment in rats. METHODS: We performed in vivo microdialysis in the hippocampus for 3-3 h of control and 4-AP treatment phase in parallel with electrophysiology measurement. Then, we analyzed the microdialysate peptidome of control and treated samples from the same subject by liquid chromatography-coupled tandem mass spectrometry. We analyzed electrophysiological and peptidomic alterations upon epileptic seizure induction by two-tailed, paired t-test. RESULTS: We detected 2540 peptides in microdialysate samples by mass spectrometry analysis; and 866 peptides-derived from 229 proteins-were found in more than half of the samples. In addition, the abundance of 322 peptides significantly altered upon epileptic seizure induction. Several proteins of significantly altered peptides are neuropeptides (Chgb) or have synapse- or brain-related functions such as the regulation of synaptic vesicle cycle (Atp6v1a, Napa), astrocyte morphology (Vim), and glutamate homeostasis (Slc3a2). CONCLUSIONS: We have detected several consequences of epileptic seizures at the peptidomic level, as altered peptide abundances of proteins that regulate epilepsy-related cellular processes. Thus, our results indicate that analyzing brain ECF by in vivo microdialysis and omics techniques is useful for monitoring brain processes, and it can be an alternative method in the discovery and analysis of CNS disease markers besides peripheral fluid analysis.

TNF-α-dependent lung inflammation upregulates PD-L1 in monocyte-derived macrophages to contribute to lung tumorigenesis.

Chronic inflammation, which is dominated by macrophage-involved inflammatory responses, is an instigator of cancer initiation. Macrophages are the most abundant immune cells in healthy lungs, and associated with lung tumor development and promotion. PD-L1 is a negative molecule in macrophages and correlated with an immunosuppressive function in tumor environment. Macrophages expressing PD-L1, rather than tumor cells, exhibits a critical role in tumor growth and progression. However, whether and how PD-L1 in macrophages contributes to inflammation-induced lung tumorigenesis requires further elucidation. Here, we found that higher expression of PD-L1 in CD11b+ CD206+ macrophages was positively correlated with tumor progression and PD-1+ CD8+ T cells population in human adenocarcinoma patients. In the urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mouse model, the infiltration of circulating CD11bhigh F4/80+ monocyte-derived macrophages (MoMs) was increased in pro-tumor inflamed lung tissues and lung adenocarcinoma. PD-L1 was mainly upregulated in MoMs associated with enhanced T cells exhaustion in lung tissues. Anti-PD-L1 treatment can reduce T cells exhaustion at pro-tumor inflammatory stage, and then inhibit tumorigenesis in IDLA. The pro-tumor lung inflammation depended on TNF-α to upregulate PD-L1 and CSN6 expression in MoMs, and induced cytokines production by alveolar type-II cells (AT-II). Furthermore, inflammatory AT-II cells could secret TNF-α to upregulate PD-L1 expression in bone-marrow driven macrophages (BM-M0). Inhibition of CSN6 decreased PD-L1 expression in TNF-α-activated macrophage in vitro, suggesting a critical role of CSN6 in PD-L1 upregulation. Thus, pro-tumor inflammation can depend on TNF-α to upregulate PD-L1 in recruited MoMs, which may be essential for lung tumorigenesis.

Inhibitory effects of vibegron, a ß3-adrenoceptor agonist, on the myogenic contractile and mechanosensitive afferent activities in an obstructed rat bladder.

To determine the role of ß3-adrenoceptor agonists on bladder sensory facilitation related to bladder myogenic contractile activities in bladder hyperactivity, we investigated the effects of vibegron, a ß3-adrenoceptor agonist, on the bladder and sensory function by evaluating cystometry and mechanosensitive single-unit afferent activities (SAAs), respectively, in a male rat model of bladder outlet obstruction (BOO). BOO was created by partial ligation of the urethra. Ten days after the surgical procedure, cystometric and SAA measurements were taken under two distinct conditions: a conscious-restrained condition, in which the bladder was constantly filled with saline, and a urethane-anesthetized condition involving an isovolumetric process with saline. For each measurement, vibegron (3 mg/kg) or its vehicle was administered intravenously after the data were reproducibly stable. In addition, the expression of ß3-adrenoceptor and substance P (SP), a sensory neuropeptide, in the bladder was further evaluated following immunohistochemical procedures. Number of non-voiding contractions (NVCs) in cystometry was decreased after vibegron-administration, which was a significant change from vehicle group. Number of microcontractions and SAAs of Aδ- and C-fibers were significantly decreased by vibegron-administration. Furthermore, ß3-adrenocepor and SP were co-expressed in the suburothelium layer of the bladder. These findings indicated that vibegron showed inhibitory effects on NVCs and microcontractions of the bladder, and SAAs of the Aδ- and C-fibers in BOO rats. The study suggested that vibegron can partly inhibit the mechanosensitive afferent transduction via Aδ- and C-fibers by suppressing bladder myogenic contractile activities in the rat bladder hyperactivity associated with BOO.

Sequence and Structure-Guided Engineering of Urethanase from Agrobacterium tumefaciens d3 for Improved Catalytic Activity.

The amidase from Agrobacterium tumefaciens d3 (AmdA) degrades the carcinogenic ethyl carbamate (EC) in alcoholic beverages. However, its limited catalytic activity hinders practical applications. Here, multiple sequence alignment was first used to predict single variants with improved activity. Afterward, AlphaFold 2 was applied to predict the three-dimensional structure of AmdA and 21 amino acids near the catalytic triad were randomized by saturation mutagenesis. Each of the mutation libraries was then screened, and the improved single variants were combined to obtain the best double variant I97L/G195A that showed a 3.1-fold increase in the urethanase activity and a 1.5-fold increase in ethanol tolerance. MD simulations revealed that the mutations shortened the distance between catalytic residues and the substrate and enhanced the occurrence of a critical hydrogen bond in the catalytic pocket. This study displayed a useful strategy to engineer an amidase for the improvement of urethanase activity, and the variant obtained provided a good candidate for applications in the food industry.

Features and application potential of microbial urethanases.

Urethanase (EC 3.5.1.75) can reduce ethyl carbamate (EC), a group 2A carcinogen found in foods and liquor. However, it is not yet commercially available. Urethanase has been detected as an intracellular enzyme from yeast, filamentous fungi, and bacteria. Based on the most recent progress in the sequence analysis of this enzyme, it was observed that amidase-type enzyme can degrade EC. All five enzymes had highly conserved sequences of amidase signature family, and their molecular masses were in the range of 52-62 kDa. The enzymes of Candida parapsilosis and Aspergillus oryzae formed a homotetramer, and that of Rhodococcus equi strain TB-60 existed as a monomer. Most urethanases exhibited amidase activity, and those of C. parapsilosis and A. oryzae also demonstrated high activity against acrylamide, which is a group 2A carcinogen. It was recently reported that urease and esterase also exhibited urethanase activity. Although research on the enzymatic degradation of EC has been very limited, recently some sequences of EC-degrading enzyme have been elucidated, and it is anticipated that new enzymes would be developed and applied into practical use. KEY POINTS: • Recently, some urethanase sequences have been elucidated • The amino acid residues that formed the catalytic triad were conserved • Urethanase shows amidase activity and can also degrade acrylamide.

Correlation between the microbial community and ethyl carbamate generated during Huzhou rice wine fermentation.

Ethyl carbamate (EC) is a potential carcinogen that is mainly produced by the spontaneous reaction between urea and ethanol during rice wine brewing. Huzhou rice wine (HZRW) is a traditional Chinese rice wine, but the correlation between its urea content and the microbial communities present during the fermentation process has not yet been evaluated. In this study, high-throughput sequencing technology was used to monitor the microbial community composition of HZRW in the different fermentation stages. The correlations between the microbial community and the physical and chemical properties and EC, urea and arginine contents were evaluated using the redundancy analysis (RDA) method. The metabolic profiles of key genes in the arginine and urea metabolic pathways were obtained via phylogenetic investigation of the communities by reconstruction of unobserved states (PICRUSt). The results showed that the fungal genera Saccharomyces, Issatchenkia, Torulaspora and Rhizopus were dominant during the fermentation of HZRW. Weissella and Acinetobacter were the dominant bacterial genera in the early stage, while Weissella, Staphylococcus, Leuconostoc and Streptophyta were the dominant bacterial genera in the late stage. Urea and arginine were positively correlated with Saccharomyces, Lactobacillus and Staphylococcus. In addition, the dominant genera of both fungi and bacteria were involved in the metabolism of arginine and urea. Finally, the relationships between the dominant microorganisms and key genes of the arginine and urea metabolic pathways were established. The obtained results are helpful in better understanding the mechanisms of metabolism of arginine and urea during rice wine fermentation and therefore improving the safety profile of rice wine.

Identification and expression of bifunctional acid urea-degrading enzyme/urethanase from Enterobacter sp. R-SYB082 and its application in degradation of ethyl carbamate in Chinese rice wine (Huangjiu).

BACKGROUND: Ethyl carbamate (EC) is a potential carcinogen existing in fermented foods such as Chinese rice wine (Huangjiu). Since urea is an important precursor of EC, the degradation of urea could be an effective way to reduce EC in foods. RESULTS: In this study, an Enterobacter sp. R-SYB082 with acid urea degradation characteristics was obtained through microbial screening. Further research isolated a new acid urea-degrading enzyme from R-SYB082 strain - ureidoglycolate amidohydrolase (UAH) - which could degrade EC directly. The cloning and expression of UAH in Escherichia coli BL21 (DE3) suggested that the activity of urea-degrading enzyme reached 3560 U L-1 , while urethanase activity reached 2883 U L-1 in the optimal fermentation condition. The enzyme had the dual ability of degrading substrate urea and product EC. The removal rate of EC in Chinese rice wine could reach 90.7%. CONCLUSION: This study provided a new method for the integrated control of EC in Chinese rice wine and other fermented foods. © 2022 Society of Chemical Industry.

The experimental study of regeneration of annulus fibrosus using decellularized annulus fibrosus matrix/poly(ether carbonate urethane)urea-blended fibrous scaffolds with varying elastic moduli.

Although tissue engineering has attracted increasing attention for the treatment of degenerative intervertebral disc disease, the biochemical properties, structural organization, and mechanical characteristics of annulus fibrosus tissue have restricted progress. Differentiation of annulus fibrosus-derived stem cells (AFSCs) can be regulated by the elasticity of substrates such as poly(ether carbonate urethane)urea (PECUU). Decellularized annulus fibrosus matrix (DAFM) has good biocompatibility and biodegradability, making it suitable for cell adhesion, proliferation, and differentiation. In this study, we used a coaxial electrospinning method to synthesize DAFM/PECUU-blended fibrous scaffolds with elasticities approximating that of native inner and outer annulus fibrosus tissue. AFSCs cultured on DAFM/PECUU-blended fibrous scaffolds exhibited increased collagen type I gene expression with increasing elasticity of the scaffold material; notably, collagen type II and aggrecan gene expression exhibited the opposite trend. Regarding extracellular matrix secretion, collagen type I content gradually increased with substrate elasticity, while collagen type II and aggrecan contents decreased. In vivo evaluations employing magnetic resonance imaging, hematoxylin and eosin staining, and immunohistochemistry indicated that DAFM/PECUU-blended fibrous scaffolds could effectively repair defects of annulus fibrosus tissue. Our findings provide a theoretical and practical basis for the development of bionic annulus fibrosus tissue that closely mimics the biological properties, mechanical function, and matrix composition of native tissue.

[Improving applicability of urease from Bacillus amyloliquefaciens JP-21 by site-directed mutagenesis].

Ethyl carbamate (EC) is a carcinogen detected in fermented foods and alcohol beverages. Excessive intake of EC is possibly harmful to health. Enzymatic degradation is one of the most effective approaches for reducing EC in fermented foods. Urease catalyzes the hydrolysis of both EC and urea. This confers urease a good application prospect in reducing EC and its precursor urea in fermented foods. Currently, degradation of EC in alcohol beverages by urease is inefficient due to its low urethanase activity and poor affinity to EC. Urease from Bacillus amyloliquefaciens JP-21 was successfully expressed in Escherichia coli at the level of 3 292 U/L urease and 227.3 U/L urethanase. Two key residues M326 and M374 were characterized that might block the binding of enzyme to EC, through simulating docking the structure of catalytic subunit UreC of urease with EC. Three mutants (M374A, M374T and M326V) of urease with improved urethanase activity were obtained by performing point saturated mutagenesis approach. Using EC as the substrate, Km values of M374A, M374T and M326V were detected to be 101.8 mmol/L, 129.5 mmol/L and 121.7 mmol/L, respectively, which were decreased by 37.47%-50.82% compared with that of the wild type urease. These mutants can degrade more than 97% of urea in rice wine and mutant M374T shows the highest degradation of EC in rice wine. EC content in rice wine was reduced from 525 µg/L to 393 µg/L by using M374T, and the EC degradation rate of it is 0.97 folds higher than that of the wild type urease. The results are of great significance for engineering the catalytic properties of urease and improving its industrial properties, and lays a good foundation for developing strategies to reducing microbial metabolic ammonia (amine) hazards in fermented foods.

Chinese Yellow Rice Wine Processing with Reduced Ethyl Carbamate Formation by Deleting Transcriptional Regulator Dal80p in Saccharomyces cerevisiae.

Ethyl carbamate (EC) is a potential carcinogen that forms spontaneously during Chinese rice wine fermentation. The primary precursor for EC formation is urea, which originates from both external sources and arginine degradation. Urea degradation is suppressed by nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. The regulation of NCR is mediated by two positive regulators (Gln3p, Gat1p/Nil1p) and two negative regulators (Dal80p/Uga43p, Deh1p/Nil2p/GZF3p). DAL80 revealed higher transcriptional level when yeast cells were cultivated under nitrogen-limited conditions. In this study, when DAL80-deleted yeast cells were compared to wild-type BY4741 cells, less urea was accumulated, and genes involved in urea utilization were up-regulated. Furthermore, Chinese rice wine fermentation was conducted using dal80Δ cells; the concentrations of urea and EC were both reduced when compared to the BY4741 and traditional fermentation starter. The findings of this work indicated Dal80p is involved in EC formation possibly through regulating urea metabolism and may be used as the potential target for EC reduction.

Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation.

Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85DUR1,2/DUR3-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85DUR1,2/DUR3-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.

Ethyl carbamate regulation and genomic expression of Saccharomyces cerevisiae during mixed-culture yellow rice wine fermentation with Lactobacillus sp.

Ethyl carbamate (EC) is a potentially carcinogenic substance present in most alcoholic beverages, especially in Chinese rice wine. Consequently, much effort has been directed at suppressing EC formation during the production of these beverages, with particular attention directed at the use of urethanase, as this enzyme can directly catalyze EC degradation. Herein, we investigated the ability of three lactic acid bacteria (Oenococcus oeni, Lactobacillus brevis, and Lactobacillus plantarum) to generate urethanase during co-cultivation with Saccharomyces cerevisiae. qPCR and transcriptomic analyses revealed that 57 genes of S. cerevisiae were significantly expressed in the presence of L. brevis, which highlighted the importance of studying urethanase-promoted EC degradation for establishing a powerful technique of EC level control. The obtained results provided deep insights into the adaptive responses of S. cerevisiae to the challenging environment of mixed-culture fermentation.

Molecular Engineering of Bacillus paralicheniformis Acid Urease To Degrade Urea and Ethyl Carbamate in Model Chinese Rice Wine.

Bacillus paralicheniformis urease (BpUrease) has been shown to be a promising biocatalyst for degrading the carcinogenic chemical ethyl carbamate (EC or urethane) in rice wine. However, low EC affinity and catalytic efficiency limit the practical application of BpUrease. In this study, we improved the EC degradation capability of BpUrease by site-saturation mutagenesis (SSM). The best variant L253P/L287N showed a 49% increase in EC affinity, 1027% increase in catalytic efficiency ( kcat/ Km), and 583% increase in half-life ( t1/2) at 70 °C. Homology modeling analysis suggest that mutation of Leu253 to Pro increased the BpUrease EC specificity by affecting the interaction between Arg339 with the catalytic residue His323, while Leu287Asn mutation benefits EC specificity and affinity by changing the interaction networks among the residues in the catalytic pocket. Our results show that the L253P/L287N variant efficiently degraded urea and EC in a model rice wine, making it a good candidate for practical application in the food industry.

Metabolic engineering of arginine permeases to reduce the formation of urea in Saccharomyces cerevisiae.

Urea is an important precursor of the harmful carcinogenic product ethyl carbamate in fermented wines. To decipher more fully the contributions of three arginine permeases, Can1p, Gap1p and Alp1p in urea formation, various engineered strains were examined for their ability to form urea. This included seven mutants with different combinations of permease deficiency and grown in both simple and more complex media, and the wild-type strain modified to overexpress the three arginine permeases. A truncated GATA transcription factor, Gln3p1-653, was also overexpressed in the arginine permease deficient mutants to determine whether the permeases have a synergistic effect on urea formation with other urea reducing modules. Additionally, in this study, transcriptional changes of four genes related to arginine metabolism and urea formation were investigated. We found that the three amino acids permeases affect urea formation mainly through the utilization of arginine in YNB medium containing the 20 common amino acids. The deletion mutant Δgap1Δcan1 showed a significant reduction (68%) in extracellular urea compared to the wild-type strain grown in YPD medium. Overexpression of a truncated Gln3p in Δgap1Δcan1 reduced the extracellular urea concentration even further (by 67%) than that in the wild-type strain and showed a synergistic effect with Δgap1Δcan1 and Δalp1Δgap1Δcan1 for extracellular reduction. Moreover, the results of this study provide a promising way to reduce urea accumulation during wine fermentation using S. cerevisiae, and present an approach to control metabolism and product formation through the regulation of amino acid permeases.

Mutant Potential Ubiquitination Sites in Dur3p Enhance the Urea and Ethyl Carbamate Reduction in a Model Rice Wine System.

Ubiquitination can significantly affect the endocytosis and degradation of plasma membrane proteins. Here, the ubiquitination of a Saccharomyces cerevisiae urea plasma membrane transporter (Dur3p) was altered. Two potential ubiquitination sites, lysine residues K556 and K571, of Dur3p were predicted and replaced by arginine, and the effects of these mutations on urea utilization and formation under different nitrogen conditions were investigated. Compared with Dur3p, the Dur3pK556R mutant showed a 20.1% decrease in ubiquitination level in yeast nitrogen base medium containing urea and glutamine. It also exhibited a >75.8% decrease in urea formation in yeast extract-peptone-dextrose medium and 41.3 and 55.4% decreases in urea and ethyl carbamate formation (a known carcinogen), respectively, in a model rice wine system. The results presented here show that the mutation of Dur3p ubiquitination sites could significantly affect urea utilization and formation. Modifying the ubiquitination of specific transporters might have promising applications in rationally engineering S. cerevisiae strains to efficiently use specific nitrogen sources.

CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae.

Enormous advances in genome editing technology have been achieved in recent decades. Among newly born genome editing technologies, CRISPR/Cas9 is considered revolutionary because it is easy to use and highly precise for editing genes in target organisms. CRISPR/Cas9 technology has also been applied for removing unfavorable target genes. In this study, we used CRISPR/Cas9 technology to reduce ethyl carbamate (EC), a potential carcinogen, which was formed during the ethanol fermentation process by yeast. Because the yeast CAR1 gene encoding arginase is the key gene to form ethyl carbamate, we inactivated the yeast CAR1 gene by the complete deletion of the gene or the introduction of a nonsense mutation in the CAR1 locus using CRISPR/Cas9 technology. The engineered yeast strain showed a 98 % decrease in specific activity of arginase while displaying a comparable ethanol fermentation performance. In addition, the CAR1-inactivated mutants showed reduced formation of EC and urea, as compared to the parental yeast strain. Importantly, CRISPR/Cas9 technology enabled generation of a CAR1-inactivated yeast strains without leaving remnants of heterologous genes from a vector, suggesting that the engineered yeast by CRISPR/Cas9 technology might sidestep GMO regulation.

Assessing the Potential Content of Ethyl Carbamate in White, Red, and Rosé Wines as a Key Factor for Pursuing Urea Degradation by Purified Acid Urease.

The ethyl carbamate (EC) content of a wine after a given temperature-time storage was theoretically predicted from the potential concentration of ethyl carbamate (PEC), as determined via an accelerated EC formation test. Such information was used to decide whether an enzymatic treatment was needed to reduce the wine urea level before bottling/aging. To this end, 6 white, red, and rosé wines, manufactured in Italy as such or enriched with urea, were tested for their PEC content either before or after enzymatic treatment using a purified acid urease preparation derived from Lactobacillus fermentum. The treatment was severely affected by the total phenolic content (TP) of the wine, the estimated pseudo-first-order kinetic rate constant for NH3 formation reducing by a factor of approximately 2000 as the TP increased from 0 to 1.64 g L(-1) . Such a sensitivity to TP was by far greater than that pertaining to a killed cell-based enzyme preparation used previously. Urea hydrolysis was successful at reducing EC concentration in wines with low levels of TP and other EC precursors.