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Saccharomyces cerevisiae engineering strain as well as preparation method, application and fermentation culture method thereof

A technology for Saccharomyces cerevisiae and engineering strains, which is applied in the field of bioengineering to achieve the effect of simple medium components

Active Publication Date: 2015-11-25
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the consumption of a large amount of carbon sources, many studies have used metabolic engineering methods to reduce the by-product ethanol, but this problem has not been completely solved so far.

Method used

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  • Saccharomyces cerevisiae engineering strain as well as preparation method, application and fermentation culture method thereof
  • Saccharomyces cerevisiae engineering strain as well as preparation method, application and fermentation culture method thereof
  • Saccharomyces cerevisiae engineering strain as well as preparation method, application and fermentation culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Construction of Example 1 Deletion Product

[0084] 1. Synthetic exogenous gene

[0085] According to the sequence of lactate dehydrogenase gene NM_174099, its DNA was artificially synthesized after optimization by J-Cat.

[0086] 2. Build missing modules

[0087] Using the genome of Saccharomyces cerevisiae BY4741 as a template, the promoters, terminators and ORF boxes of pyruvate decarboxylase genes PDC1, PDC5 and PDC6 and acetaldehyde dehydrogenase genes ADH1 and ADH4 were amplified. The amplification primer sequences of each part are as follows:

[0088] Table 1 Amplification Primer Sequence

[0089]

[0090]

[0091]

[0092] According to the PDC5-related primer sequences in Table 1, the promoter, terminator, nutritional marker URA3 and ORF box part of the pyruvate decarboxylase gene PDC5 were amplified, and the above products were connected together by PCR reaction to obtain the deletion module ΔPDC5 as figure 2 shown.

[0093] The promoter, termina...

Embodiment 2

[0097] Embodiment 2: Construction of gene knockout strain

[0098] 1. Inoculate BY4741 strain in YPD medium and culture overnight at 30°C.

[0099] 2. Transfer the culture solution of the above overnight strain to 5ml of fresh YPD to make the initial OD 600 =0.1, continue culturing at 30°C for 4-6 hours to make the culture medium OD 600 Reach 0.4-0.6.

[0100] 3. Transformation: The deletion module ΔPDC5 obtained above was transformed into competent cells made of BY4741 by the lithium acetate transformation method, and then spread on SC-Ura plates after transformation and cultured at 30°C.

[0101] 4. Genome extraction verification: pick the transformant, extract the genome for PCR verification label, BY4741 is used as a negative control, and the band size is correct, indicating that the homologous recombination of the missing module is correct and completed.

[0102] 5. Pop off the nutritional marker Ura3: the correct transformant will be verified and marked on FoA, and th...

Embodiment 3

[0120] Example 3 Molecular biology verification

[0121] The extraction method of the yeast genome refers to the yeast genetics experiment guide, and the primers used in the molecular biology verification of Example 2 are as follows:

[0122] 1. After transforming the missing module, the PCR verification primers for Ura3 were not removed:

[0123] The primers for the verification of deletion module ΔPDC5 recombination were upstream p5_cku: 5'-TTTCAGCTCTTTCAAGTTCCTC-3' (SEQ ID NO: 45), downstream P_p5_dw: 5'-CATGAGTTTTATGTTAATTAGCTTATTTGTTCTTCTTGTTATTGTATTGTGTTGTTC-3' (SEQ ID NO: 46), and upstream U_p5_ACT: 5'-TCCTTAAGAT3ATGCTCGAATACTAAGAG (SEQ ID NO: 47), downstream p5_ckd1: 5'-TAGACTGGTCTTTGGGTAGTGTAGG-3' (SEQ ID NO: 48).

[0124] The primers for the verification of deletion module ΔPDC6 recombination were upstream p6_cku: 5'-ATGTCCATTGGAATATGCAGA-3' (SEQ ID NO: 49), downstream P_p6_dw: 5'-GTTTGAGTACACTACTAATGGCTTATTTGTTGGCAATATGTTTTTGCTATATTACGTG-3' (SEQ ID NO: 50), and ups...

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Abstract

The invention relates to the technical field of bioengineering, and in particular relates to a saccharomyces cerevisiae engineering strain as well as a preparation method, application and a fermentation culture method thereof. The PDC1, PDC5, PDC6, ADH1 and ADH4 genes of the saccharomyces cerevisiae engineering strain are knocked out, and an LDH gene is inserted into the saccharomyces cerevisiae engineering strain. The saccharomyces cerevisiae engineering strain provided by the invention does not contain an ethanol metabolism main route, a lot of glucose can be used for fermentation so as to accumulate lactic acid, and the strain can grow quickly by using ethanol.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an engineering strain of Saccharomyces cerevisiae and its preparation method, application, and fermentation cultivation method. Background technique [0002] L-lactic acid is an organic acid refined from corn starch through biological fermentation. It is a colorless and clear viscous liquid, and its aqueous solution shows an acidic reaction. It can be freely mixed with water, ethanol or ether, and insoluble in chloroform. Because of its left-handed characteristics, it has good biological compatibility, can be compatible with mammals, can directly participate in human metabolism, without any side effects, and is widely used in food, medicine and other fields: [0003] Food industry: It is mainly used in food processing industries such as candy and beverages (such as beer, wine and lactic acid beverages), as a sour agent and taste regulator, and is known as an absolutely sa...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N1/36C12P7/56C12R1/865
Inventor 元英进白雪李炳志王靖宇
Owner TIANJIN UNIV
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