Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Industrial rice wine yeast metabolic engineering bacteria with low-yield urea and building method thereof

A rice wine yeast and metabolic engineering technology, applied in fungi, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems affecting the growth and fermentation ability of Saccharomyces cerevisiae, low transformation efficiency and other problems

Inactive Publication Date: 2014-04-09
JIANGNAN UNIV
View PDF6 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The wine and sake yeast transformed by the Van Vuuren team are diploid industrial yeast strains. Due to the lack of available defective selection markers, the yeast was transformed by co-transfection with the plasmid, and the transformation efficiency was low, and the highly expressed component (URA3 -PGK1p-DUR1,2-PGK1t-URA3) is used for the transformation of haploid strains, and the obtained engineered bacteria are uracil-deficient, which will affect the growth and fermentation ability of Saccharomyces cerevisiae

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Industrial rice wine yeast metabolic engineering bacteria with low-yield urea and building method thereof
  • Industrial rice wine yeast metabolic engineering bacteria with low-yield urea and building method thereof
  • Industrial rice wine yeast metabolic engineering bacteria with low-yield urea and building method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of uracil-deficient yeast engineering strain Δura3

[0045] 1. Construction of URA3 gene knockout module

[0046] Extract the genomic DNA of the parental strain (see the instruction manual of the MiniBEST Universal Genomic DNA Extraction Kit kit for specific operation methods); use the genomic DNA as a template, and use Prime STAR DNA Polymerase to amplify the upstream of the URA3 gene using primer pairs P1 / P2 and P3 / P4, respectively The homology arm (URA3L) and the downstream homology arm (URA3R), and then use P1 / P4 primers to fuse URA3L and URA3R by fusion PCR to obtain the URA3 gene knockout module (URA3L-URA3R) (the specific operation method and reaction system of fusion PCR Please refer to Prime STAR DNA Polymerase instructions for preparation).

[0047] The amplification procedure for each primer pair is as follows:

[0048] (1) P1 / P2 and P3 / P4: Pre-denaturation at 95°C for 4 minutes, (denaturation at 98°C for 10 s, annealing at 52°C for 1...

Embodiment 2

[0064] Example 2 Construction of diploid industrial yellow rice wine yeast engineering bacteria with high expression of urea amidase gene (DUR1, 2)

[0065] 1. Construction of high expression module DUR1,2L-URA3-PGK1p-DUR1,2R containing PGK1p strong promoter and URA3 gene

[0066] For a schematic diagram of the build, see image 3 . First, using the genomic DNA of industrial rice wine yeast as a template, Prime STAR DNA Polymerase was used to amplify the URA3 gene, PGK1p strong promoter, DUR1,2 initiation Homology arm before the codon (DUR1,2L), homology arm after the start codon of DUR1,2 (DUR1,2R). Then, using the equimolar four fragments as a template, the above four fragments were fused by landing PCR without primers. The PCR reaction system is shown in Table 2:

[0067] Table 225μL PCR reaction system

[0068]

[0069] The PCR amplification program is as follows: pre-denaturation at 95°C for 4 min, then enter the PCR amplification program, denaturation at 98°C for...

Embodiment 3

[0084] Embodiment 3 yellow rice wine fermentation experiment

[0085] The diploid yeast engineered bacterium obtained in Example 2 is carried out rice wine fermentation experiment together with parent bacterial strain to investigate the ability of engineered bacterium to reduce urea and EC content, concrete operation steps are as follows:

[0086] (1) Soak rice

[0087] Take 50g of glutinous rice, add water to soak the rice layer for about 10cm, and soak for 48 hours at room temperature;

[0088] (2) Cooking

[0089] Cook the soaked rice under normal pressure, and keep it for 25 minutes after a large amount of steam comes out, until the grains are uniform, there is no white heart inside, cooked but not sticky, soft but not rotten;

[0090] (3) Bibimbap

[0091] Cool the rice in a ventilated place, spread it to about 27°C, add 1.5g of cooked wheat koji, 7g of raw wheat koji, and 85g of water;

[0092] (4) Pre-fermentation

[0093] The fermented mash was added to the activa...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses industrial rice wine yeast metabolic engineering bacteria with low-yield urea. The building method of the engineering bacteria comprises the following steps: building URA3 gene knockout component, respectively transforming a-type and alpha-type haploid rice wine yeasts, so as to obtain a uracil defect type strain; building a strong promoter containing PGK1p and a high-expression component of a URA3 gene; respectively transforming the a-type and alpha-type haploid rice wine yeasts; integrating into a strain genome by homologous recombination; inserting the PGK1p strong promoter before an initiation codon of a DUR1,2 gene to obtain a prototrophic haploid engineering strain for high expression of the DUR1,2 gene; fusing into diploid again, so as to obtain the industrial rice wine yeast metabolic engineering bacteria with low-yield urea. The engineering bacteria disclosed by the invention are prototrophic, the growth and fermentation abilities are very nearly the same as those of a parent strain, the content of urea and EC in a fermentation liquor can be obviously reduced, and no exogenous gene is led in. Therefore, the industrial rice wine yeast metabolic engineering bacteria are high in biosecurity and have good industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and fermentation, and in particular relates to a metabolic engineering of industrial rice wine yeast with low production of urea and ethyl carbamate (EC) and a construction method thereof. Background technique [0002] Ethyl Carbamate (EC for short), commonly known as urethane, is a harmful by-product produced during the fermentation of rice wine and has genotoxicity. With the increase of people's consumption of alcoholic beverages and the growing rice wine industry, the existence of EC is not only a health hazard for consumers, but also hinders the pace of internationalization of rice wine. Therefore, it is particularly important to control the content of EC in rice wine. [0003] Urea is the main precursor of EC in fermented wine. Most of EC in rice wine comes from the spontaneous reaction product of urea and ethanol, and the main source of urea in fermented mash is produced by yeast met...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/19C12N15/81
Inventor 陆健吴殿辉陈坚李晓敏谢广发申超
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com