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Method for producing lactic acid

一种乳酸、生物质的技术，应用在生物化学设备和方法、裂解酶、肽等方向，能够解决未满足改进工艺等问题，达到高产量、高生产率、降低生产成本的效果

Inactive Publication Date: 2019-05-03
SYCONIUM LACTIC ACID GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0023] However, although several attempts have been made to improve lactic acid producing yeast, there remains an unmet need to provide an improved process for regenerating large quantities of pure isomeric lactic acid without the need for costly purification processes

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  • Method for producing lactic acid
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  • Method for producing lactic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0257] Example 1: Characterization of the fermentation performance of Saccharomyces cerevisiae (CBS7962) strains at different glucose and lactic acid concentrations and pH.

[0258] The S. cerevisiae CBS7962 strain was grown in two stages in bioreactors and shake flasks. Use containing 1.8g / lYNB, 5g / l (NH 4 ) 2 SO 4 and 20g / l glucose medium for the inoculum incubation stage. Cells stored in glycerol vials stored at -80°C were used to inoculate 50 ml of preinoculum medium with an optical density at 600 nm (OD600) of 0.05. After 24 hours of growth, cells were harvested and used as inoculum during the culture phase in shake flasks and in bioreactors (Eppendorf DASGIPDASbox4, Germany) with working volumes of 50 ml and 700 ml, respectively. Table 1 and Table 2 list the medium composition and fermentation parameters of the culture stage. All cultures were carried out at 30°C; the shake flasks were incubated in an orbital shaker set at 180 rpm; potassium hydrogen phthalate buffe...

Embodiment 2

[0263] Example 2: Construction of S. cerevisiae strains lacking pyruvate decarboxylase (PDC1 , PDC5 and PDC6) genes using a split-maker deletion method.

[0264] Allelic knockout of PDC1, PDC5, and PDC6 was facilitated by a split marker deletion method using kanamycin and hygromycin as selection markers. The primer sequences used for deletion and validation primers are listed in Table 3. Two constructs were designed for deletion of one of the alleles. Each construct contained flanks of the target gene, followed by LoxP sites, and about half of one of the selectable markers. Flanking regions and LoxP sites were included in primers used to amplify part of the selectable markers for kanamycin and hygromycin from pPM2aK21 and pPM2a_hphR, respectively. Two PCR constructs containing target gene flanking regions, a LoxP site and part of one of the selectable markers were transformed into the CBS7962 strain. Transformation was performed according to the LiAc / PEG / ss-DNA protocol (ht...

Embodiment 3

[0267] Example 3: Construction of Saccharomyces cerevisiae strains with inactive pyruvate decarboxylase (PDC1, PDC5 and PDC6) genes: The pdc gene was knocked out using the CRISPR-cas9 system.

[0268] The CRISPR-cas9 system, which uses RNA-guided endonuclease activity to induce double-strand DNA breaks (dsbs) at target sites and repairs dsbs with double-strand (ds) DNA homologous oligonucleotides containing stop codons, is used to inactivate pdc gene. Transformation was performed according to the LiAc / PEG / ss-DNA protocol (http: / / home.cc.umanitoba.ca / ~gietz / method.html). Using golden gate assembly, the endonuclease cas9 was constructed in a centromeric plasmid under the TEF1 promoter and CYC1 terminator (Engler et al., 2008). Guide RNAs (gRNAs) for the PDC1 , PDC5 and PDC6 genes were identified using ChopChop (https: / / chopchop.rc.fas.harvard.edu / ). A 20 bp gRNA was constructed to contain the sequence of the hammerhead (HH) ribozyme and the sequence of the hepatitis D virus (H...

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Abstract

The present invention provides a method for producing lactic acid in a recombinant yeast cell culture using glucose as carbon source comprising a first, seed fermentation stage to produce biomass wherein the yeast is cultivated in a culture medium at a p H of 5 to 7, followed by a second, a production fermentation stage with biomass from the seed fermentation to produce lactic acid, wherein the yeast is cultivated in a culture medium at low p H using a yeast strain that is engineered to have lactate dehydrogenase (LDH) activity and optionally has decreased or knocked- out pyruvate decarboxylase (PDC) activity.

Description

technical field [0001] The present invention provides a method for the production of lactic acid in recombinant yeast cell culture using glucose as a carbon source, comprising the production of biomass in a first seed fermentation stage, wherein the yeast is cultured in a medium with a pH of 5 to 7, followed by A second productive fermentation stage for the production of lactic acid from seed-fermented biomass in which the yeast is grown in a medium at low pH using a yeast strain engineered to have lactate dehydrogenase (LDH) activity or It has lactate dehydrogenase (LDH) activity and reduced or knocked out pyruvate decarboxylase (PDC) activity. Background technique [0002] Lactic acid is listed as a drug candidate in the top 30 chemical building blocks of sugars by the U.S. Department of Energy, showing significant market growth. The global lactic acid market was estimated at 714,200 tons in 2013 and is projected to expand to 1,960,100 tons by 2020, growing at a compound ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/18C12N15/52C12P7/56
CPCC12N1/18C12N15/52C07K14/395C12N9/0006C12N9/88C12P7/56C12Y101/01027C12Y401/01001
Inventor M·阿斯克R·科普拉姆D·马塔诺维赫M·绍尔
Owner SYCONIUM LACTIC ACID GMBH
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