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Application of chromatin regulatory factor Ahc1p in reducing urea accumulation of saccharomyces cerevisiae

A regulatory factor, the technology of Saccharomyces cerevisiae, applied in the field of microbial genetics and molecular biology, to achieve the effect of reducing accumulation, improving utilization rate and reducing waste

Active Publication Date: 2022-03-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the fermentation process, yeast cells are regulated by NCR, and the preferential nitrogen source will inhibit the expression of urea utilization-related enzyme genes, resulting in the accumulation of high-concentration urea in the cells.

Method used

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  • Application of chromatin regulatory factor Ahc1p in reducing urea accumulation of saccharomyces cerevisiae
  • Application of chromatin regulatory factor Ahc1p in reducing urea accumulation of saccharomyces cerevisiae
  • Application of chromatin regulatory factor Ahc1p in reducing urea accumulation of saccharomyces cerevisiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Construction of the bacterial strain overexpressing AHC1 in embodiment 1

[0063] The pY26-AHC1 overexpression plasmid was constructed using pY26-GPD-TEF as the vector. Use primers AHC1 (F) and AHC1 (R) to amplify the AHC1 gene sequence (nucleotide sequence as shown in SEQ ID NO.2) from Saccharomyces cerevisiae S288C-ura3, use primers pY26 (F) and pY26 (R) PCR linearization of the pY26 vector. Then, the gene fragment and the linearized plasmid were connected by a one-step cloning method, and transformed into JM109 strain. The plasmid was extracted, sequenced correctly, and then transformed into the S288C-ura3 strain by lithium acetate method. The positive transformants were screened by colony PCR to obtain a recombinant strain overexpressing AHC1.

[0064] The pY26-GPD-TEF empty plasmid was transformed into S288C-ura3 strain as a control.

[0065] All primers and gene sequences are listed in Table 5.

[0066] Table 5 Primer Sequence

[0067]

Embodiment 2

[0068] Example 2: Effect of overexpression of AHC1 on urea accumulation

[0069] The recombinant strain constructed in Example 1 and the control strain (S288C-pY26) were activated by culturing on a shaker at 200 rpm at 30°C for 18 hours (OD after activation 600 =1.2), they were inoculated in the single nitrogen source medium with the preferred nitrogen source asparagine as the only nitrogen source, the single nitrogen source medium with the neutral nitrogen source arginine as the only nitrogen source, and the non-preferred nitrogen source In a single nitrogen source medium in which proline is the only nitrogen source, ferment at 30°C for 48 hours, and detect the accumulation of urea. Test results such as figure 1 As shown, when asparagine, arginine and urea were used as the sole nitrogen source, the urea produced by S288C-pY26-AHC1 within 48 hours was 51.55%, 54.50% and 37.21% lower than that of the control strain, respectively.

Embodiment 3

[0070] Example 3: Effect of Overexpression of AHC1 on Amino Acid Utilization

[0071] The recombinant bacterial strain constructed in Example 1 and the control bacterial strain were respectively activated by culturing at 200 rpm at 30°C for 18 hours (OD after activation 600 = 1.2), inoculated in a mixed medium containing 21 kinds of commonly used amino acids, fermented at 30°C for 48 hours, and tested the utilization rate of the strains to 21 kinds of nitrogen sources. Test results such as figure 2 As shown, compared with the control strain, Saccharomyces cerevisiae S288C-pY26-AHC1 overexpressing AHC1 significantly increased the utilization rate of Pro, Tyr and Arg by 413.28%, 339.04% and 32.83%, and the utilization rate of Val, Thr, Gly and Leu The utilization rate increased by 16.96%, 15.44%, 12.69% and 10.07% respectively. These results suggest that overexpression of AHC1 increases utilization of multiple nitrogen sources. This is of great significance for the applicati...

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Abstract

The invention discloses application of a chromatin regulatory factor Ahc1p in reducing urea accumulation of saccharomyces cerevisiae, and belongs to the technical field of microbial inheritance and molecular biology. According to the invention, a chromatin regulatory factor Ahc1p gene sequence in the saccharomyces cerevisiae is connected to a high-copy plasmid, and is converted into the saccharomyces cerevisiae for free expression, so that the urea accumulation amounts of the saccharomyces cerevisiae over-expressing the Ahc1p gene in a culture medium taking asparagine, arginine or proline as a sole nitrogen source are respectively reduced by 51.55%, 54.50% and 37.21%. The utilization rate of various non-preferred nitrogen sources is increased, so that the accumulation of harmful nitrogen metabolites is reduced, the utilization rate of the non-preferred nitrogen sources in the raw materials is expected to be greatly increased, and the waste of the raw materials is reduced.

Description

technical field [0001] The invention relates to the application of chromatin regulatory factor Ahc1p in reducing urea accumulation in Saccharomyces cerevisiae, and belongs to the technical field of microbial genetics and molecular biology. Background technique [0002] In the production process of traditional fermented products, such as rice wine, due to the incomplete metabolism of nitrogen-containing compounds during the fermentation process, a large amount of nitrogen sources are wasted and harmful substances such as ethyl carbamate in fermented foods accumulate. Urethane has neurotoxicity and strong carcinogenicity, and is rated as a Class 2A hazardous substance by the International Cancer Research Institute (IARC). Urea is one of the important precursors of ethyl carbamate, and about 70%-90% of ethyl carbamate in rice wine is formed by the spontaneous reaction of urea and ethanol. [0003] Most of the urea produced in the fermentation process comes from the metabolism ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/31C12N1/19C07K14/395C12G3/02C12R1/865
CPCC07K14/395C12N15/81C12G3/02Y02E50/10
Inventor 余世琴周景文陈宇曾伟主陈坚
Owner JIANGNAN UNIV