高通量筛选诱变菌株降低黄酒发酵氨基甲酸乙酯前体积累

【目的】氨基甲酸乙酯是发酵食品中普遍存在的一种潜在危害物。黄酒中氨基甲酸乙酯的前体主要是尿素和乙醇。本研究通过高通量筛选策略降低黄酒发酵过程中尿素的积累,从而降低氨基甲酸乙酯积累。【方法】以一株黄酒生产工业菌株酿酒酵母XZ-11为研究对象,采用ARTP诱变和高通量筛选策略,获得尿素积累量较低菌株。使用实时定量PCR技术检测氮代谢中尿素代谢和转运相关基因(DUR1,2和DUR3)的变化。【结果】筛选得到一株尿素高效稳定性利用菌株5-11C。其尿素积累量比酿酒酵母XZ-11降低了50.6%。实时定量荧光PCR结果表明,与尿素代谢和转运相关的基因(DUR1,2和DUR3)表达量分别提高了3.3和2.2倍。【结论】高通量筛选策略可以用于降低黄酒生产过程中氨基甲酸乙酯前体尿素的含量。由于未采用基因工程手段,避免了可能的法规问题,消费者易于接受,在发酵食品行业具有较好的应用前景。     

The urea transporter DUR3 contributes to rice production under nitrogen‐deficient and field conditions

Nitrogen is one of the most important elements for plant growth, and urea is one of the most frequently used nitrogen fertilizers worldwide. Besides the exogenously‐supplied urea to the soil, urea is endogenously synthesized during secondary nitrogen metabolism. Here, we investigated the contribution of a urea transporter, DUR3, to rice production using a reverse genetic approach combined with localization studies. Tos17 insertion lines for DUR3 showed a 50% yield reduction in hydroponic culture, and a 26.2% yield reduction in a paddy field, because of decreased grain filling. Because shoot biomass production and shoot total N was not reduced, insertion lines were disordered not only in nitrogen acquisition but also in nitrogen allocation. During seed development, DUR3 insertion lines accumulated nitrogen in leaves and could not sufficiently develop their panicles, although shoot and root dry weights were not significantly different from the wild‐type. The urea concentration in old leaf harvested from DUR3 insertion lines was lower than that in wild‐type. DUR3 promoter‐dependent β‐glucuronidase (GUS) activity was localized in vascular tissue and the midribs of old leaves. These results indicate that DUR3 contributes to nitrogen translocation and rice yield under nitrogen‐deficient and field conditions.     

Reduced production of ethyl carbamate for wine fermentation by deleting <Emphasis Type="Italic">CAR1</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.     

Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)⁺ mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)⁺ mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.     

Mutant isolation of non-urea producing sake yeast by positive selection

Urea is reported to be a main precursor in wine and sake (Japanese rice wine) of ethyl carbamate (ECA), a suspected carcinogen. We constructed an arginase-deficient mutant (Δcar1/Δcar1) from a diploid sake yeast, Kyokai no. 9, using a gene disruption method (Kitamoto, K. et al., Appl. Environ. Microbiol., 57, 301, 1991). The car1 mutant thus constructed enabled us to brew sake containing no urea or ECA. In spite of their superior characteristics, industrial use of mutants for sake brewing has so far been difficult because guidelines for recombinant DNA utilization in the food and beverages industry have not yet been established. Using the genetically engineered car1 mutant, we have developed a new medium for the positive selection of car1 mutants. Many arginase-deficient mutants could be easily isolated from not only a laboratory haploid strain (X2180-1A), but also sake yeasts (Kyokai no. 9 and Kyokai no 10) and wine yeasts (Geisenheim 74 and Eperney). Sake with no urea could be brewed using the car1 mutants, and no ECA was detected in the resulting sake even after heat treatment (hi-ire) and storage.     

Rapamycin treatment results in GATA factor-independent hyperphosphorylation of the proline utilization pathway activator in Saccharomyces cerevisiae

Treatment of Saccharomyces cerevisiae cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation. Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes. The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin. Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment. Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p. The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene PUT1 were also examined. We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced PUT1 expression. Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased PUT1 expression. Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation. A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source.     

Genetic Basis of Variations in Nitrogen Source Utilization in Four Wine Commercial Yeast Strains

The capacity of wine yeast to utilize the nitrogen available in grape must directly correlates with the fermentation and growth rates of all wine yeast fermentation stages and is, thus, of critical importance for wine production. Here we precisely quantified the ability of low complexity nitrogen compounds to support fast, efficient and rapidly initiated growth of four commercially important wine strains. Nitrogen substrate abundance in grape must failed to correlate with the rate or the efficiency of nitrogen source utilization, but well predicted lag phase length. Thus, human domestication of yeast for grape must growth has had, at the most, a marginal impact on wine yeast growth rates and efficiencies, but may have left a surprising imprint on the time required to adjust metabolism from non growth to growth. Wine yeast nitrogen source utilization deviated from that of the lab strain experimentation, but also varied between wine strains. Each wine yeast lineage harbored nitrogen source utilization defects that were private to that strain. By a massive hemizygote analysis, we traced the genetic basis of the most glaring of these defects, near inability of the PDM wine strain to utilize methionine, as consequence of mutations in its ARO8, ADE5,7 and VBA3 alleles. We also identified candidate causative mutations in these genes. The methionine defect of PDM is potentially very interesting as the strain can, in some circumstances, overproduce foul tasting H₂S, a trait which likely stems from insufficient methionine catabolization. The poor adaptation of wine yeast to the grape must nitrogen environment, and the presence of defects in each lineage, open up wine strain optimization through biotechnological endeavors.     

Allantoin catabolism influences the production of antibiotics in Streptomyces coelicolor

Purines are a primary source of carbon and nitrogen in soil; however, their metabolism is poorly understood in Streptomyces. Using a combination of proteomics, metabolomics, and metabolic engineering, we characterized the allantoin pathway in Streptomyces coelicolor. When cells grew in glucose minimal medium with allantoin as the sole nitrogen source, quantitative proteomics identified 38 enzymes upregulated and 28 downregulated. This allowed identifying six new functional enzymes involved in allantoin metabolism in S. coelicolor. From those, using a combination of biochemical and genetic engineering tools, it was found that allantoinase (EC 3.5.2.5) and allantoicase (EC 3.5.3.4) are essential for allantoin metabolism in S. coelicolor. Metabolomics showed that under these growth conditions, there is a significant intracellular accumulation of urea and amino acids, which eventually results in urea and ammonium release into the culture medium. Antibiotic production of a urease mutant strain showed that the catabolism of allantoin, and the subsequent release of ammonium, inhibits antibiotic production. These observations link the antibiotic production impairment with an imbalance in nitrogen metabolism and provide the first evidence of an interaction between purine metabolism and antibiotic biosynthesis.     

Cloning and nucleotide sequence of the urea amidolyase gene from Candida utilis

Urea amidolyase (EC 3.5.1.45) is an important multi-functional enzyme for the degradation of urea. The urea amidolyase gene from Candida utilis CA(u)-37 (DUR1,2^c) was cloned by plaque hybridization, and the nucleotide sequences of DUR1, 2^c and its flanking regions were determined. DUR1, 2^c was found to be composed of 5,490 base pairs and 1,830 amino acid residues. Using Edman degradation of the purified enzyme, it was revealed that the amino-terminal residue (methionine) was processed for maturation. A TATA-box like sequence was found 112 bases upstream from the translation start site (ATG). The site of the poly (A) tail was found 54 bases downstream from the translation stop site (TGA), since cDNA of DUR1, 2^c was synthesized from mRNA and sequenced. The nucleotide sequences of the urea amidolyase gene from Saccharomyces cerevisiae and DUR1, 2^c were very similar to each other (65.3%), as were the deduced amino acid sequences (67.2%). The molecular weight of DUR1, 2^c was calculated to be 200,700. This value corresponded to the result obtained from SDS-polyacrylamide gel electrophoresis of the purified enzyme. The enzyme functions in a dimeric form. Three important regions were found in the amino acid sequence of urea amidolyase through the homology search. It was predicted that each region was equivalent to the active site of allophanate hydrolase, that of urea carboxylase, and the biotin-binding site. This was verified by deletion analysis of the DUR1, 2^c gene in S. cerevisiae. The function of the upstream region of the C. utilis gene is also discussed.