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Improved yeast strains for organic acid production

A yeast strain and organic acid technology, applied in fermentation, fungi, etc., can solve problems that have not been described

Inactive Publication Date: 2011-05-04
UNIV DEGLI STUDI DI MILANO BICOCCA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] However, the effect of hexose uptake on organic acid production with microorganisms has never been described

Method used

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  • Improved yeast strains for organic acid production
  • Improved yeast strains for organic acid production
  • Improved yeast strains for organic acid production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1: Construction of Saccharomyces cerevisiae (GRF18U and CEN.PK) strains expressing (over)expressing HXT1 or HXT7 genes: Cell growth, glucose consumption and ethanol production of transformants and control strains.

[0133] Saccharomyces cerevisiae HXT1 and HXT7 genes were PCR amplified using genomic DNA extracted from the GRF18U strain as a template.

[0134] The oligonucleotides used for amplification were as follows:

[0135] 5'HXT1 (SEQ ID NO.: 1)

[0136] 5′-AAA ATC ATG AAT TCA ACT CCC GAT CTA-3’Tm: 58.9

[0137] 3'HXT1 (SEQ ID NO.: 2)

[0138] 5′-AGC TTG TTT AGT TTA TTT CCT GCTG AAA-3′Tm: 59.3

[0139] 5'HXT7 (SEQ ID NO.: 3)

[0140] 5′-A AAA ATG TCA CAA GAC GCT GCT ATT GCA-3′Tm: 62.4

[0141] 3'HXT7 exit (SEQ ID NO.: 4)

[0142] 5′-ATA TAT TAA AAA CGT ATT TAC TTT TCA AGT-3′Tm: 54.2

[0143] 3'HXT7 (SEQ ID NO.: 5)

[0144] 5′-AGT GTC GAC AAA TAA TTT GGT GCT GAA CAT-3′ Tm: 61.0

[0145] All amplifications use the following procedure:

[0146]

...

Embodiment 2

[0155] Example 2: Construction of Saccharomyces cerevisiae (GRF18U and CEN.PK) strains expressing heterologous LDH activity from Lactobacillus plantarum and (over)expressing HXT1 or HXT7 genes: cell growth, glucose consumption, lactic acid and ethanol production.

[0156] S. cerevisiae strains (GRF18U and CEN.PK) that had been transformed with p022HXT1 and p022HXT7 expression vectors were further transformed with a plasmid called Ycp111bTLDH. The plasmid was obtained by inserting the LDH gene under the control of the Z. bayeria TPI promoter in the basic S. cerevisiae centromeric plasmid Ycplac111 (LEU2 marker, ACX75457). In order to obtain the plasmid, the Lactobacillus plantarum LDH gene was PCR-amplified in advance and subcloned into the Escherichia coli vector pSTBlue to obtain the pSTplLDH plasmid (Microb Cell Fact. January 30, 2006; 5:4.Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate ...

Embodiment 3

[0159] Example 3: Construction of Saccharomyces cerevisiae (CEN.PK) strains expressing heterologous LDH activity from Lactobacillus plantarum and (over)expressing HXT1 or HXT7 genes; cell growth, glucose consumption, lactic acid and ethanol production of transformants and control strains .

[0160] In this example, the lactic acid and ethanol production of the CEN.PK strain transformed with multiple copies of L. plantarum LDH was compared to that obtained with one extra copy of the HXT1 or HXT7 gene.

[0161] Bacterial lactate dehydrogenase was excised from the aforementioned pSTplLDH with EcoRI and inserted into similarly cleaved and dephosphorylated plasmid pYX212 (basic Saccharomyces cerevisiae multicopy expression plasmid pYX212, LEU2 marker, R&D Systems, Inc., Wiesbaden , D), the expression plasmid p212lpLDH was obtained. Independent transformants derived from yeast transformation were cultured in shake flasks in minimal medium (YNB, 1.34% w / V YNB from Difco Laboratories...

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Abstract

The present invention relates to the production of organic acids with yeasts that overexpress at least one sugar transporter. The yeast might express further genes related to the production of the desired organic acid. The organic acid is produced by cultivation of the yeast overexpressing a sugar transporter in an adequate culture medium, whereupon the desired organic acid is accumulated in the culture medium and subsequently purified to the desired degree by techniques known in the art.

Description

[0001] The present invention relates to the production of organic acids by yeast. More specifically, the invention relates to organic acid producing yeast and to methods of producing organic acids by yeast overexpressing one or more hexose transporters. Background of the invention [0002] A variety of organic acids are widely used in the food and pharmaceutical industries, and organic acids have attracted more and more attention as new building block materials in the chemical industry (Werpy, T. and G. Petersen, Top Value Added Chemicals From Biomass.2004, US Department of Energy: Oak Ridge TN). In particular, they offer a reasonable alternative to petroleum-derived and thus limited building block materials if they are produced by environmentally friendly fermentation strategies. Examples of such organic acids include lactic acid, citric acid, itaconic acid and succinic acid. [0003] Microorganisms useful in such methods include bacteria, filamentous fungi and yeasts. How...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12P7/40C12P7/56
CPCC12N1/16C12P7/40C12P7/56
Inventor D·波罗P·布兰多尔迪M·绍尔
Owner UNIV DEGLI STUDI DI MILANO BICOCCA
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