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

Construction and application of an engineered strain of Saccharomyces cerevisiae producing l-malic acid

A technology of Saccharomyces cerevisiae and engineering bacteria, applied in fermentation, enzymes, fungi, etc., can solve problems such as heavy metabolic burden, overexpression of large fragment genes, and long fermentation cycle

Active Publication Date: 2018-10-16
JIANGNAN UNIV
View PDF1 Cites 2 Cited by
  • 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

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
  • Construction and application of an engineered strain of Saccharomyces cerevisiae producing l-malic acid
  • Construction and application of an engineered strain of Saccharomyces cerevisiae producing l-malic acid
  • Construction and application of an engineered strain of Saccharomyces cerevisiae producing l-malic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0036] (1) Using the cDNA of A.flavus ATCC13697 as a template, use primer 1 (sequence such as SEQ ID NO.1) and primer 2 (sequence such as SEQ ID NO.2) to extract pyruvate carboxylase from A.flavus ATCC13697 Gene (NCBI accession numble: XM_002377040,3582bp) carries out PCR amplification, utilizes primer 3 (sequence such as SEQ ID NO.3) and primer 4 (sequence such as SEQ ID NO.4) to amplify malate dehydrogenase gene (NCBI accession numble : XM_002378178,996bp), utilize primer 5 (sequence such as SEQ ID NO.5) and primer 6 (sequence such as SEQ ID NO.6) to carry out PCR amplification malate transporter gene (NCBI accession numble: 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: ...

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...

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

PropertyMeasurementUnit
wavelengthaaaaaaaaaa
Login to View More

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

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
Patent Type & Authority Patents(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
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