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Recombinant yeast producing 3-hydroxypropionic acid and method for producing 3-hydroxypropionic acid using the same

A technology for recombining yeast and yeast, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve problems such as low 3-HP productivity and complex 3-HP metabolic pathways

Active Publication Date: 2017-08-01
SK INNOVATION CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] There are various documents and patents related to methods of genetically modifying Saccharomyces cerevisiae in various yeast strains to produce 3-HP, but in the case of Saccharomyces cerevisiae, since 3-HP passes through [pyruvate→acetaldehyde → acetic acid → acetyl coenzyme A → malonyl coenzyme A → malonate semialdehyde → 3-HP] pathway production, there is the problem that the metabolic pathway for producing 3-HP is complex and the productivity of 3-HP is relatively low (US2010 / 0248233A1 ; Y. Chen et al., Metabolic Engineering, 22:104-109, 2014)

Method used

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  • Recombinant yeast producing 3-hydroxypropionic acid and method for producing 3-hydroxypropionic acid using the same
  • Recombinant yeast producing 3-hydroxypropionic acid and method for producing 3-hydroxypropionic acid using the same
  • Recombinant yeast producing 3-hydroxypropionic acid and method for producing 3-hydroxypropionic acid using the same

Examples

Experimental program
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Embodiment 1

[0104] Example 1 Selection of host yeast strains based on tolerance to 3-HP

[0105] An essential feature of 3-HP-producing organisms is a good tolerance to high concentrations of 3-HP, which enables product accumulation during fermentation with minimal loss of strain performance. A large, diverse set of 718 wild-type yeast strains was screened to identify those strains that can tolerate high concentrations of 3-HP at low pH and can grow and metabolize glucose under these conditions ( Table 12).

[0106] The entire panel of strains was initially screened for acid tolerance using a number of agar plate and liquid medium microtiter plate based growth assays. Subsets of strains were then evaluated for their ability to tolerate large amounts of 3-HP at low pH in shake flask cultures. None of these screening methods in isolation is a perfect indicator of 3-HP tolerance in industrial settings, but the combination of many methods provides a comprehensive and robust means of identif...

Embodiment 2

[0138] Example 2 Bioinformatics Genome Mining to Obtain Pathway Enzyme Candidates

[0139] Homology-based database searches were performed to identify candidate enzymes for specific functions. In each case, the database is searched with multiple query sequences. In addition, members of related protein families were retrieved from Uniprot / SwissProt based on InterPro domain annotations. Homology-based searches were performed against the Uniprot (SwissProt and TrEMBL) and GenBank protein databases (nr, pat, and env_nr) using blastp, and against the GenBank nucleotide databases (tsa_nt, env_nt, and pat) using tblastn. Sequences with E values ​​less than 1e-30 were extracted; however, in some cases additional analyzes were performed for sequences with E values ​​less than 1e-80. Using the query sequence as a guide in translation, the nucleotide hits were translated into protein sequences using GeneWise. The task of translating long ACC sequences was too difficult for GeneWise, t...

Embodiment 3

[0160] The measurement of embodiment 3 enzyme activity

[0161] ACC Spectrophotometric Enzyme Assay

[0162] The spectrophotometric ACC assay is a coupled assay in which the product produced by the ACC reaction is further consumed in a reaction requiring the cofactor NAD(P)H, the oxidation of the cofactor NAD(P)H can be monitored spectrophotometrically.

[0163] Kroeger et al. (2011, Anal. Biochem. 411:100-105) describe a conjugation assay in which malonyl-CoA produced by ACC1 is passed through purified malonyl-CoA reductase (MCR) in the presence of NADPH as coenzyme. In the reaction of the factor, it is further converted into malonate semialdehyde. ACC activity is measured by following NADPH oxidation.

[0164] Diacovich et al. (2002, J. Biol. Chem. 277:31228-31236) combined ADP conversion (the hydrolysis product of ATP used as a cofactor in the ACC reaction) with the pyruvate kinase reaction requiring ADP, which The reaction is further coupled to the formation of pyruvate...

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Abstract

Provided are a recombinant yeast producing 3-hydroxypropionic acid (3-HP) and a method for producing 3-HP using the same, more particularly, a recombinant yeast producing 3-HP, comprising an exogenous AADH gene; an endogenous or exogenous ACC gene; an exogenous MCR gene; and an exogenous HPDH gene, and producing 3-HP through [pyruvic acid to acetaldehyde to acetyl-coA to malonyl-coA to malonate semialdehyde to 3-HP] biosynthesis pathway, and a method for producing 3-HP using the same.

Description

technical field [0001] The present invention relates to a recombinant yeast producing 3-hydroxypropionic acid (3-HP) and a method for producing 3-HP using it, more specifically, to a recombinant yeast producing 3-HP, which comprises an exogenous gene encoding AADH, encoding The endogenous gene or exogenous gene of ACC, the exogenous gene encoding MCR and the exogenous gene encoding HPDH, and through [pyruvate (Pyruvate) → acetaldehyde → acetyl-CoA → malonyl-CoA → malonate half Aldehyde → 3-HP] biosynthesis pathway to produce 3-HP, and a method for producing 3-HP using the recombinant yeast. Background technique [0002] 3-HP (3-hydroxypropionic acid, C3) is an isomer of lactic acid (2-hydroxypropionic acid), which has a carboxylic acid group and a hydroxyl group at both ends, so it is capable of being converted into various chemicals (such as 1, 3-propanediol, acrylic acid, acrylamide, polymers, etc.) are useful materials. In fact, 3-HP was selected by the US Department of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N1/16C12N1/18C12P7/42C12R1/865C12R1/72C12R1/645
CPCC12N1/16C12N1/18C12N9/0006C12N9/0008C12N9/93C12N15/52C12P7/42C12Y101/0103C12Y101/01031C12Y101/01059C12Y101/01061C12Y101/01298C12Y102/0101C12Y102/01075C12Y604/01002C12N15/81C12Y101/01C12Y208/04001
Inventor 朴重珉朴宰演朴佑赞李相珉徐荣彬欧佳·梅里亚科维史度恩·欧蒂康利·安德鲁勒门·梅里亚罗胡恩·劳拉朱赫恩·宝拉
Owner SK INNOVATION CO LTD
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