Yeast for producing tyrosine and hydroxytyrosine and construction method

A technology for producing hydroxyphenol and constructing method, applied in the field of bioengineering, can solve problems such as yeast that have not been retrieved, achieve important economic value and social benefits, good safety, and improve the effect of yield

Active Publication Date: 2018-11-06
SHANDONG HENGLU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] At the same time, no biosynthesis technology has been found for the production of tyrosol and hydroxytyrosol using yeast, especially Saccharomyces cerevisiae

Method used

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  • Yeast for producing tyrosine and hydroxytyrosine and construction method
  • Yeast for producing tyrosine and hydroxytyrosine and construction method
  • Yeast for producing tyrosine and hydroxytyrosine and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Construction of recombinant expression plasmid pJFE3-PcAAS-ADH

[0061] The amino acid sequences of SEQ ID NO.1 and SEQ ID NO.2 are codon-optimized according to the codon preference of the host Saccharomyces cerevisiae, and after obtaining the optimized nucleotide sequences corresponding to SEQ ID NO.1 and SEQ ID NO.2, Perform gene synthesis. Primers were selected, and the target gene was amplified with KOD FX DNA polymerase from TOYOBO Company. After agarose gel electrophoresis to verify the correct size of the band, the band was cut out, and the gene fragment was recovered with an OMEGA gel extraction kit. PCR amplification primers are as follows:

[0062] The sequence of PcAAS-F is SEQ ID NO.9; the sequence of PcAAS-R is SEQ ID NO.10; the sequence of adh-F is SEQ ID NO.11; the sequence of adh-R is SEQ ID NO.12;

[0063] With the E.coli strain containing the GBdir+pSC101-BAD-ETgA-tet plasmid, pick a single colony on a plate containing tetracycline resistance and tr...

Embodiment 2

[0080] Construction of the pdc1 knockout cassette

[0081] Using the Saccharomyces cerevisiae genome as a template, use primers PDC1UF / PDC1UR and PDC1DF / PDC1DR to amplify the upstream and downstream 500bp homology arms of the pdc1 fragment gene, use primers G418F / G418R to amplify the G418 resistance gene fragment, and use fusion PCR to construct pdc1 Knockout cassette sequencing verification.

[0082] Primer sequence:

[0083] The sequence of PDC1UF is SEQ ID NO.13; the sequence of PDC1UR is SEQ ID NO.14; the sequence of G418F is SEQ ID NO.15; the sequence of G418R is SEQ ID NO.16; the sequence of PDC1DF is SEQ ID NO.17; The sequence of PDClDR is SEQ ID NO.18.

Embodiment 3

[0085] Construction of tyrA expression cassette

[0086] Using the Saccharomyces cerevisiae genome as a template, use primers PDC1F-YZ / PDC1UF1 and the upstream 500bp homology arm, use the pdc1 knockout cassette constructed in Example 2 as a template, and use primers G418F1 / PDCIR-YZ to amplify tyrA and pdc1 downstream 500bp Fragment, using the Escherichia coli BL-21 (DE3) genome as a template, using primers tyrAF1 / tyrAR1 to amplify the tyrA fragment of the gene, using the method of fusion PCR to construct the tyrA expression cassette and verify it by sequencing.

[0087] Primer sequence:

[0088] The sequence of PDC1F-YZ is SEQ ID NO.19; the sequence of PDC1UF1 is SEQ ID NO.20; the sequence of TYRAF1 is SEQ ID NO.21; the sequence of TYRAR1 is SEQ ID NO.22; the sequence of G418F1 is SEQ ID NO. 23; the sequence of PDCIR-YZ is SEQ ID NO.24.

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Abstract

The invention relates to a yeast for producing tyrosine and hydroxytyrosine and a construction method, and belongs to the technical field of preparation of organic compounds. The construction method comprises the following steps of introducing the sequences PcAAS and ADH into the yeast BY4741, so as to obtain the recombinant yeast PcAAS-ADH for producing the tyrosine; introducing a pdc1 gene knockoff cassette and a tyrA expression cassette into the recombinant yeast PcAAS-ADH, so as to obtain the recombinant yeast PcAAS-ADH-deltapdc1-tyrA for producing the tyrosine; introducing the DNA (deoxyribonucleic acid) sequence of HpaBC into the recombinant yeast PcAAS-ADH-deltapdc1-tyrA, so as to obtain the recombinant yeast PcAAS-ADH-HpaBC-deltapdc1-tyrA for producing the hydroxytyrosine. The tyrosine or hydroxytyrosine biological synthesis route is constructed in the yeast BY4741, so that the output of the tyrosine or hydroxytyrosine is increased.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a genetically recombined yeast with high tyrosol production, a Saccharomyces cerevisiae capable of heterologously synthesizing hydroxytyrosol, and a construction method of the yeast. Background technique [0002] Tyrosol is a natural antioxidant derived from olive oil and a derivative of phenylethyl alcohol. Also known as salidroside aglycon, it is the main medicinal active ingredient of rhodiola rosea, and is the precursor of salidroside and hydroxytyrosol. It can protect cells from oxidative damage. It is a phenolic compound with important industrial value. Tyrosol and its derivatives are the synthetic precursors of various organic compounds. Tyrosol can be used in pharmaceuticals. Hydroxytyrosol, a derivative of tyrosol, is a strong antioxidant and a variety of physiological and medical functions. Hydroxytyrosol has stronger antioxidant properties than tyrosol. At the sa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/22C12R1/86
CPCC12N9/0006C12N9/88C12N15/81C12P7/22C12Y401/01025C12N15/52C12Y101/01001
Inventor 方诩郭伟韩丽娟
Owner SHANDONG HENGLU BIOTECH CO LTD
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