Establishment and application of brewing yeast engineering bacterium strain for producing L-malic acid

A technology of Saccharomyces cerevisiae and engineering bacteria, applied in the direction of enzymes, fermentation, fungi, etc., can solve the problems of long fermentation cycle, overexpression of large fragment genes, and low bacterial concentration

Active Publication Date: 2016-03-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are still several key problems in the use of Saccharomyces cerevisiae to accumulate malic acid: 1) long fermentation cycle; 2) low production intensity; 3) low bacterial concentration and slow growth of strains; 4) heterologous expression of eukaryotic genes such as molds 5) Overexpression of large fragment genes, heavy metabolic burden, etc.

Method used

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  • Establishment and application of brewing yeast engineering bacterium strain for producing L-malic acid
  • Establishment and application of brewing yeast engineering bacterium strain for producing L-malic acid
  • Establishment and application of brewing yeast engineering bacterium strain for producing L-malic acid

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Effect test

Embodiment 1

[0035] Example 1: Construction of Saccharomyces cerevisiae engineering strains producing malic acid

[0036] (1) Using the cDNA of A.flavusATCC13697 as a template, the pyruvate carboxylase gene (NCBIaccessionnumble: XM_002377040, 3582bp) to carry out PCR amplification, utilize primer 3 (sequence such as SEQIDNO.3) and primer 4 (sequence such as SEQIDNO.4) to amplify malate dehydrogenase gene (NCBIaccessionnumble: XM_002378178,996bp), utilize primer 5 (sequence such as SEQIDNO. .5) and primer 6 (sequence such as SEQ ID NO.6) for PCR amplification of the malate transporter gene (NCBI accessionnumble: XM_002376564, 1197bp);

[0037] The primer sequences are as follows:

[0038] Primer 1: 5'-CGGGATCCATGGCGGCTCCGTTTCGT-3'

[0039] Primer 2: 5'-AACTGCAGTTGCTTACGCTTTGACGAT-3'

[0040] Primer 3: 5'-ATTTGCGGCCGCATGGTCAAAGCTGCGGTACT-3'

[0041] Primer 4: 5'-GAAGATCTTCAAAGCTTTGGTGGTGGGTT-3'

[0042] Primer 5: 5'-CGGAATTCATGTTCAATAACGAACACC-3'

[0043] Primer 6: 5'-CCGCTCGAGCTAATCAG...

Embodiment 2

[0053] Embodiment 2: utilize Saccharomyces cerevisiae to produce L-malic acid

[0054] Inoculate the engineered strains stored in the glycerol tube on the slant of the YNB medium, take a ring to the seed medium (100mL / 500mL Erlenmeyer flask), and cultivate it at 30°C and 200r / min for 48h, then inoculate with 10% inoculum (V / V ) to inoculate the fermentation medium (100mL / 500mL Erlenmeyer flask), the temperature is 30°C, the rotation speed is 800rpm, the ventilation rate is 1.5vvm, the pH is adjusted to 5.0 by 8mM KOH, and the fermentation time is 84h. Measured by high performance liquid chromatography (HPLC): the malic acid output of the genetically engineered bacteria (W1101) of the present invention is 27.3 g / L, while the starting strain (WT) accumulates a small amount of malic acid. The yield of malic acid relative to glucose was 0.41mol / mol.

[0055] Table 1 product output (unit: g / L)

[0056]

[0057] Advantages of the present invention: 1) Compared with the reported...

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Abstract

The invention discloses establishment and application of a brewing yeast engineering bacterium strain for producing L-malic acid, and belongs to the field of fermentation engineering. Genes of pyruvic carboxylase (Afpyc), malic dehydrogenase (Afmdh) and malic acid transport protein (Afmae) coming from Aspergillus flavus ATCC13697 are excessively and dissociatively expressed in the bacterium strain S.cerevisiae tTAM(delta)ura3(delta)trpl high in pyruvic acid yield, the malic acid accumulation path is established, and the bacterium W1101 is obtained. The bacterium strain is used for producing L-malic acid through fermentation; after fermentation is conducted for 84 h, the malic acid yield is 27.3 g / L; an original starting bacterium strain does not accumulate malic acid, the metabolism path of aspergillus flavus of the high-yield L-malic acid bacterium strain is successfully applied to brewing yeast, and a new strategy is provided for establishing the high-yield L-malic acid bacterium strain.

Description

technical field [0001] The invention relates to the construction and application of a Saccharomyces cerevisiae engineering bacterium producing L-malic acid, belonging to the field of fermentation engineering. Background technique [0002] Malic acid, also known as dihydroxysuccinic acid, is an important four-carbon platform compound and is listed as one of the 12 potential compounds by the US Department of Energy. It is an important intermediate product of the TCA biological cycle. It has a special pleasant sour taste and is easily absorbed and utilized by the human body. Therefore, it is widely used in the food industry as a food sour agent with excellent performance and functional food. At the same time, it has the functions of anti-oxidation and inhibiting oil rancidity, and is used in cosmetics, medicine, and chemical industries. [0003] At present, the production methods of malic acid mainly include: biocatalytic method, two-step fermentation method and one-step ferme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/46C12R1/865
CPCC07K14/38C12N9/0006C12N9/93C12P7/46C12Y101/01037C12Y604/01001
Inventor 刘立明王元彩陈修来陈瑞东董晓翔高聪胡贵鹏郭亮
Owner JIANGNAN UNIV
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