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Acid-resistant saccharomyces cerevisiae and application thereof in preparation of organic acid

A technology of Saccharomyces cerevisiae and organic acid, which is applied in the field of microorganisms, can solve the problems of resource waste environment, increase the cost and process of product separation and purification, pollution, etc., and achieve the effects of saving costs, reducing separation and purification processes, and super acid resistance

Active Publication Date: 2021-08-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At the same time, in the prior art, the method of alkali neutralization increases the cost and process of product separation and purification on the one hand. It will cause waste of resources and pollution of the environment

Method used

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  • Acid-resistant saccharomyces cerevisiae and application thereof in preparation of organic acid
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  • Acid-resistant saccharomyces cerevisiae and application thereof in preparation of organic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Evolution and screening of Saccharomyces cerevisiae cells

[0047] Specific steps are as follows:

[0048] (1) Using Saccharomyces cerevisiae CEN.PK2-1C as the starting strain, malic acid was used as the selection pressure in the first stage, and different concentrations of malic acid were added exogenously, and Saccharomyces cerevisiae tolerant to lower pH was screened by directed evolution mutant strain. By adding different concentrations of malic acid, the pH is controlled to decrease step by step (starting from pH 6.0, the amount of exogenous malic acid added is 4.6g / L), and after the cells in each pH level have evolved to be able to grow, flat culture is carried out for enrichment (The target is the OD of the cells at 48h at this pH 600 Stability is reached, and there is no significant increase in the first two times; the purpose is to increase the number of mutant cells and make cell growth more stable at this pH) before proceeding to the evolution of...

Embodiment 2

[0053] Example 2: Analysis of cell stability of Saccharomyces cerevisiae CTPth-2

[0054] In order to verify the stability of the obtained Saccharomyces cerevisiae CTPth-2 under low pH conditions, the specific steps are as follows:

[0055] After Saccharomyces cerevisiae CTPth-2 was continuously passaged for 50 times, Saccharomyces cerevisiae CTPth-2 and the starting strain Saccharomyces cerevisiae CEN.PK2-1C were inoculated into YPD medium respectively. In the case of growth comparison. The added concentration (g / L) of exogenous citric acid is 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and the pH values ​​are 7.0, 3.85, 3.29, 3.09, 2.78 , 2.59, 2.43, 2.31, 2.24, 2.18; samples were taken every 12 hours until 72 hours, and the growth status of the strain was determined. The results are shown in Table 1, wherein Saccharomyces cerevisiae CTPth-2 is abbreviated as CTPth-2; the starting strain Saccharomyces cerevisiae CEN.PK2-1C is abbreviated as CEN.PK2-1C.

[0056] Table 1: OD of ...

Embodiment 3

[0059] Embodiment 3: the synthesis of malic acid

[0060] In order to better verify the potential of Saccharomyces cerevisiae CTPth-2 as a chassis cell to produce organic acids, in the present invention, taking malic acid as an example, the target product is synthesized through metabolic modification.

[0061] Specific steps are as follows:

[0062] 1. Construction of recombinant strains:

[0063] (1) Chemically synthesize the nucleotide sequence encoding malate dehydrogenase MDH3ΔSKL (malate dehydrogenase MDH3 removes the last three amino acids SKL), the nucleotide sequence encoding pyruvate carboxylase pyc2, and the nucleus encoding malate transporter SpMAE1 Nucleotide sequence (wherein, the nucleotide sequence encoding the MDH3 enzyme is shown in SEQ ID NO.2, the nucleotide sequence encoding the PYC2 enzyme is shown in SEQ ID NO.4, the encoding SpMAE1 enzyme The nucleotide sequence is shown in SEQ ID NO.6);

[0064] (2) Construction of the recombinant vector:

[0065] T...

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Abstract

The invention discloses acid-resistant saccharomyces cerevisiae and application thereof in preparation of organic acid. The invention provides a strain of saccharomyces cerevisiae. The saccharomyces cerevisiae is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC M 2021258, and the preservation date is March 19, 2021; the saccharomyces cerevisiae CTPth-2 is obtained through directed evolution screening, and the lowest tolerance pH value is about 2.32, which is also the lowest tolerance pH value of the saccharomyces cerevisiae reported at present; and in order to verify the potential of acid-resistant chassis cell saccharomyces cerevisiae CTPth-2 for producing organic acids, malic acid is taken as an example, a cytoplasm pathway for malic acid synthesis is strengthened, malic acid transporter protein from Schizosaccharomyces pombe is subjected to exogenous expression, an engineering strain CTPM3 is constructed, shake flask fermentation is adopted under the condition that a neutralizer is not added, and the fermentation yield of the malic acid reaches 16.5 g / L.

Description

technical field [0001] The invention relates to an acid-resistant Saccharomyces cerevisiae and its application in the preparation of organic acids, belonging to the technical field of microorganisms. Background technique [0002] Organic acids, especially short-chain organic acids, have been widely used in food, biomedicine, cosmetics, detergents, polymers and textiles in recent years. Short-chain organic acids are low molecular weight organic compounds with one or more acidic groups, most typically carboxylic acid groups. According to the number of carbon atoms, short-chain organic acids can be divided into carbon two (C2), carbon three (C3), carbon four (C4), carbon five (C5) and carbon six (C6) organic acids. Generally, C3 organic acids include propionic acid, pyruvic acid, lactic acid and 3-hydroxypropionic acid. C4 organic acids include malic, fumaric, succinic and butyric acids. C5 organic acids include itaconic acid, alpha-ketoglutaric acid, glutaric acid, and xylu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/04C12N9/88C12N15/81C07K14/39C12P7/46C12P7/52C12P7/40C12P7/56C12P7/50C12P7/44C12P19/02C12P7/48C12R1/865
CPCC12N9/0006C12N9/88C07K14/39C12N15/81C12P7/46C12P7/52C12P7/40C12P7/56C12P7/50C12P7/44C12P19/02C12P7/48C12Y101/01037C12Y401/01031Y02E50/10
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰孙利
Owner JIANGNAN UNIV
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