YEAST PRODUCING TYROSOL OR HYDROXYTYROSOL, AND CONSTRUCTION METHODS THEREOF

Abstract

The technical field of preparation of organic compounds, and particularly to yeasts producing tyrosol or hydroxytyrosol and construction methods thereof. PcAAS and ADH-encoding DNA sequences are introduced into the yeast strain BY4741, to obtain a PcAAS-ADH recombinant yeast producing tyrosol. A PDC1 knockout cassette and a TyrA expression cassette are introduced into the PcAAS-ADH recombinant yeast to obtain a PcAAS-ADH-ΔPDC1-TyrA recombinant yeast producing tyrosol. A HpaBC encoding DNA sequence is introduced into the PcAAS-ADH-ΔPDC1-TyrA recombinant yeast, to obtain a PcAAS-ADH-HpaBC-ΔPDC1-TyrA recombinant yeast producing hydroxytyrosol. The construction of a tyrosol or hydroxytyrosol biosynthesis pathway in the yeast strain BY4741 enhances the production of tyrosol or hydroxytyrosol.

Claims

1. A yeast producing tyrosol, constructed through a process comprising specifically the step of introducing a tyrosine decarboxylase encoding DNA sequence derived from Petroselinum crispum and an alcohol dehydrogenase encoding DNA sequence derived from Enterobacteriaceae into the yeast strain BY4741, to obtain a PcAAS-ADH recombinant yeast.

2. The yeast producing tyrosol according to claim 1, wherein the tyrosine decarboxylase has an amino acid sequence as shown in SEQ ID NO. 1; the DNA sequence encoding the tyrosine decarboxylase is as shown in SEQ ID NO. 2, which expresses the amino acid sequence as shown in SEQ ID NO. 1; the alcohol dehydrogenase has an amino acid sequence as shown in SEQ ID NO. 3; and the DNA sequence encoding the alcohol dehydrogenase is as shown in SEQ ID NO. 4, which expresses the amino acid sequence as shown in SEQ ID NO. 3.

3. The yeast producing tyrosol according to claim 1, wherein a PDC1 knockout cassette is introduced into the PcAAS-ADH recombinant yeast according to claim 1, to obtain a PcAAS-ADH-ΔPDC1 recombinant yeast, wherein the PDC1 knockout cassette is constructed through a process comprising specifically the steps of: by using the genome of the yeast strain BY4741 as a template, amplifying an upstream and a downstream 500 bp homology arm of the PDC1 fragment with primers, amplifying the G418 resistant gene fragment with primers, and fusing the upstream and downstream 500 bp homology arms and the G418 resistant gene fragment, to obtain the PDC1 knockout cassette.

4. The yeast producing tyrosol according to claim 3, wherein a tyrA expression cassette is introduced into the PcAAS-ADH-ΔPDC1 recombinant yeast according to claim 3, to obtain a PcAAS-ADH-ΔPDC1-TyrA recombinant yeast, wherein the TyrA expression cassette is constructed through a process comprising specifically the steps of: by using the genome of the yeast strain BY4741 as a template, amplifying an upstream 500 bp homology arm with primers; by using the PDC1 knockout cassette according to claim 3 as a template, amplifying the TyrA fragment with primers; and fusing the upstream 500 bp homology arm and the TyrA fragment to construct the TyrA expression cassette.

5. A yeast producing hydroxytyrosol, wherein a cluster of 4-hydroxyphenylacetic hydroxylase encoding DNA sequences derived from Escherichia coli are introduced into the PcAAS-ADH-ΔPDC1-TyrA recombinant yeast according to claim 4, to obtain a PcAAS-ADH-HpaBC-ΔPDC1-TyrA recombinant yeast producing hydroxytyrosol.

6. The yeast producing hydroxytyrosol according to claim 5, wherein a cluster of amino acid sequences of 4-hydroxyphenylacetic hydroxylase comprise sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 7; and a cluster of 4-hydroxyphenylacetic hydroxylase encoding DNA sequences comprise sequences as shown in SEQ ID NO. 6 and SEQ ID NO. 8, which express the amino acid sequence as shown in SEQ ID NO. 5 and the amino acid sequence as shown in SEQ ID NO. 7 respectively.

7. A method for constructing a yeast producing tyrosol, comprising specifically the steps of: 1) inserting PcAAS and ADH-encoding DNA sequences into an expression vector, to construct a vector-PcAAS-ADH recombinant expression plasmid; 2) constructing a PDC1 knockout cassette according to claim 3; 3) constructing a TyrA expression cassette through a process comprising specifically the steps of: by using the genome of the yeast strain BY4741 as a template, amplifying an upstream 500 bp homology arm with primers; by using the PDC1 knockout cassette obtained in Step 2) as a template, amplifying the TyrA fragment with primers; and fusing the upstream 500 bp homology arm and the TyrA fragment to construct the TyrA expression cassette; and 4) inserting the PDC1 knockout cassette obtained in Step 2) and the TyrA expression cassette obtained in Step 3) into the vector-PcAAS-ADH recombinant expression plasmid obtained in Step 1), to obtain a PcAAS-ADH-ΔPDC1-TyrA plasmid, and inserting the PcAAS-ADH-ΔPDC1-TyrA plasmid into the yeast strain BY4741, to obtain a recombinant yeast strain PcAAS-ADH-ΔPDC1-TyrA; wherein the expression vector in Step 1) is preferably pJFE3, pUC19, pδBLE2.0, pGK series vectors, or pXP318; and further preferably, the vector is pJFE3.

8. A method for constructing a yeast producing hydroxytyrosol, comprising specifically the steps of: 1) synthesizing a HpaB and a HpaC encoding gene to construct a HpaBC expression cassette; and 2) introducing the HpaBC expression cassette into the recombinant PcAAS-ADH-Δpdc1-tyrA yeast, to obtain a recombinant PcAAS-ADH-Δpdc1-tyrA-HpaBC yeast.

9. Use of the yeast producing tyrosol according to claim 1 and the method for constructing a yeast producing tyrosol comprising specifically fermenting with the yeast producing tyrosol to obtain tyrosol, wherein a medium for the fermentation is one or a mixture of glucose, fructose, sucrose, glucose and tyrosine.

10. Use of the yeast producing hydroxytyrosol according to claim 5 and the method for constructing a yeast producing hydroxytyrosol, comprising specifically fermenting with the yeast producing hydroxytyrosol to obtain hydroxytyrosol, wherein the medium for the fermentation is one or a mixture of glucose, fructose, sucrose, glucose and tyrosine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The accompanying drawings of this specification constituting a part of this application are used to provide further understanding of this application. The exemplary embodiments and descriptions thereof of this application are intended to explain this application, and do not constitute improper restriction to this application

[0053] FIG. 1 is a schematic diagram showing a pathway for synthesizing tyrosol and hydroxytyrosol with glucose or tyrosine as a substrate.

DETAILED DESCRIPTION

[0054] It should be noted that the following detailed descriptions are exemplary and are intended to provide further understanding of this application. Unless otherwise indicated, meanings of all technical and scientific terms used in this specification are the same as that usually understood by a person skilled in the technical field to which this application belongs.

[0055] It should be noted that, terms used herein are merely used to describe specific implementations, and are not intended to limit exemplary implementations according to this application. As used herein, unless otherwise specified in the context clearly, the singular forms are intended to include the plural forms as well. In addition, it should be further understood that the terms “include”, and/or “comprise”, when used in this specification, indicate the presence of features, steps, operations, devices, components, and/or combinations thereof.

[0056] The present invention is further described below with reference to embodiments: [0057] Table 1 shows abbreviations and full names of terms used herein

TABLE-US-00001 Abbreviation Full name Glucose PEP Phosphoenolpyruvate PREP Prephenate 2HxThPP 2-(alpha-Hydroxyethyl) thiamine diphosphate 4HPP 4-Hydroxyphenylpyruvate Tyr L-Tyrosine 4HPAA 4-Hydroxyphenylacetaldehyde TYR-OL 4-Hydroxyphenylethanol

Example 1

[0058] Construction of pJFE3-PcAAS-ADH Recombinant Expression Plasmid

[0059] The amino acid sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2 were codon-optimized according to the codon preference of the host Saccharomyces cerevisiae, to obtain optimized nucleotide sequences corresponding to SEQ ID NO. 1 and SEQ ID NO. 2 for gene synthesis. Amplification using selected primers by KOD FXDNA polymerase available from TOYBO gave the target genes. The bands verified to have a correct size by agarose gel electrophoresis was cut, and the gene fragments were recovered using the OMEGA gel extraction kit. Primers for PCR amplification

[0060] PcAAS-F sequence as shown in SEQ ID NO. 9; PcAAS-R sequence as shown in SEQ ID NO. 10; adh-F sequence as shown in SEQ ID NO. 11; and adh-R sequence as shown in SEQ ID NO. 12.

[0061] Single colonies of Escherichia coli strain containing the GBdir+pSC101-BAD-ETgA-tet plasmid on a plate coated with tetracycline as a resistance screening agent were picked into an EP tube containing 1 mL LB liquid medium (sterilized, containing 4 ug/mL tetracycline), and incubated overnight at 30° C. and 200 rpm. The target gene fragments were amplified by high-fidelity KOD FX DNA polymerase and each fragment was ensured to have a 50 bp homologous sequence with the adjacent fragment. The PCR products were subjected to gel electrophoresis and then recovered using a DNA fragment extraction kit. The DNA concentration was determined. Then the fusion fragments were amenable to RED/ET recombination as follows:

[0062] (1) 40 μL of the culture incubated overnight was pipetted to 1 mL of fresh LB medium containing 4 μg/mL tetracycline and shaken at 30° C. and 200 rpm for 2 h.

[0063] (2) The fragments were ligated using T4 DNA polymerase. The reaction system is shown in Table 2:

TABLE-US-00002 TABLE 2 Reaction system Component Amount (uL) DNA fragment 500 ng each 10 × NE Buffer 2 2 100 × BSA Buffer 0.2 T4 DNA polymerase 0.13 dd H.sub.2O q.s. to 20 μl In total 20

[0064] The above reagents were added to a PCR tube and reacted under conditions as shown in Table 3.

TABLE-US-00003 TABLE 3 Reaction conditions Reaction temperature Reaction time 25° C. 60 min 75° C. 20 min 50° C. 30 min  4° C. ∞

[0065] (3) 40 μL of 10% L-arabinose solution was added to the bacterial solution in Step 1, and shaken at 37° C. and 200 rpm for 40 min.

[0066] (4) The system obtained after reaction in Step 2 was desalted for 40 min using VSWP01300 MF-Millipore white MCE hydrophilic filter membrane with a smooth surface having a pore size of 0.025 um and a diameter of 13 mm.

[0067] (5) The bacterial solution obtained in Step 3 was centrifuged at 9000 rpm for 1 min. The supernatant was discarded, and the pellet was added to 500 μL of sterilized ddH.sub.2O to re-suspend the bacterial solution. The bacterial solution was then centrifuged at 9000 rpm for 1 min. The supernatant was discarded. After the process was repeated twice, the supernatant was discarded, and the pellet was added to 50 μL of sterilized ddH.sub.2O to re-suspend the bacterial solution and then allowed to stand on ice.

[0068] (6) The desalted solution obtained in Step 4 was uniformly mixed with the treated bacterial solution obtained in Step 5, and allowed to stand on ice for 1 min.

[0069] All the mixture was pipetted and subjected to electroporation (parameters: 1350 V, 200 Ω, 25 mA, 1 uF).

[0070] (8) 1 mL of fresh LB liquid medium (sterilized, containing no resistance screening agent) was added to the electroporation cuvette, beaten evenly, pipetted into an EP tube, and incubated at 37° C. and 200 rpm or 1 h.

[0071] (9) 50 μL of the bacterial solution was coated onto an LB plate containing 50-100 ug/ml Amp and incubated at 37° C. overnight.

[0072] (10) Single clones were picked up for verification by PCR and sequencing.

Example 2

[0073] Construction of PDC1 Knockout Cassette

[0074] By using the genome of Saccharomyces cerevisiae as a template, an upstream and a downstream 500 bp homology arm of the PDC1 fragment were amplified by using the primers PDC1UF/PDC1UR and PDC1DF/PDC1DR, and the G418 resistant gene fragment was amplified by using the primers G418F/G418R. A PDC1 knockout cassette was constructed by fusion PRC, and verified by sequencing.

[0075] Primer Sequences:

[0076] PDC1UF sequence as shown in SEQ ID NO. 13; PDC1UR sequence as shown in SEQ ID NO. 14; G418F sequence as shown in SEQ ID NO. 15; G418R sequence as shown in SEQ ID NO. 16; PDC1DF sequence as shown in SEQ ID NO. 17; and PDC1DR sequence as shown in SEQ ID NO. 18.

Example 3

[0077] Construction of TyrA Expression Cassette

[0078] By using the genome of Saccharomyces cerevisiae as a template and using the primers PDC1F-YZ/PDC1UF1 and the upstream 500 bp homology arm, and by using the pdc1 knockout cassette constructed in Example 2 as a template and using the primers G418F1/PDCIR-YZ, tyrA and the downstream 500 bp fragment of pdc1 were amplified. By using the genome of Escherichia coli BL-21(DE3) as a template and using the primers tyrAF1/tyrAR1, the tyrA gene fragment was amplified. A tyrA expression cassette was constructed by fusion PRC, and verified by sequencing.

[0079] Primer Sequences:

[0080] PDC1F-YZ sequence as shown in SEQ ID NO. 19; PDC1UF1 sequence as shown in SEQ ID NO. 20; TyrAF1 sequence as shown in SEQ ID NO. 21; TyrAR1 sequence as shown in SEQ ID NO. 22; G418F1 sequence as shown in SEQ ID NO. 23; and PDCIR-YZ sequence as shown in SEQ ID NO. 24.

Example 4

[0081] Construction of HpaBC Expression Cassette

[0082] The amino acid sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 7 were codon-optimized according to the codon preference of the host Saccharomyces cerevisiae, to obtain optimized nucleotide sequences as shown in SEQ ID NO. 6 and SEQ ID NO. 8 corresponding to SEQ ID NO. 5 and SEQ ID NO. 7 for gene synthesis. A HpaBC expression cassette was constructed according to the method described in Example 1.

Example 5

[0083] Construction of Microbial Strains Heterologously Synthesizing Tyrosol, with Saccharomyces cerevisiae as an Example:

[0084] The strain obtained in Example 1 was inoculated in a liquid LB medium, and incubated at 37° C. and 200 rpm for 14 h, and then extracted with the OMEGA plasmid extraction kit D6943-01 to obtain the pJFE3-PcAAS-ADH recombinant expression plasmid. Saccharomyces cerevisiae BY4741 was transformed by the PEG/LiAc method. Single clones were screened by using the URA selective medium, and the plasmid was extracted. The clones were verified by PCR using the primers YZ1F and YZ1R to obtain the PcAAS-ADH strain. The PDC1 knockout cassette obtained in Example 2 and the TyrA expression cassette obtained in Example 3 were introduced into the PcAAS-ADH strain to obtain the PcAAS-ADH-ΔPDC1-TyrA strain. On the basis of the PcAAS-ADH-ΔPDC1-TyrA strain, the HpaBC expression cassette constructed in Example 5 was introduced into the PcAAS-ADH-ΔPDC1-TyrA strain by the PEG/LiAc method, to obtain the PcAAS-ADH-ΔPDC1-TyrA-HpaBC strain.

[0085] The YZ1F sequence is as shown in SEQ ID NO. 25; and the YZ1R sequence is as shown in SEQ ID NO. 26.

Example 6

[0086] Fermentation with Microorganisms Synthesizing Tyrosol, with Saccharomyces cerevisiae as an Example:

[0087] Single clones were picked up from a plate containing the PcAAS-ADH strain or PcAAS-ADH-ΔPDC1-TyrA strain producing tyrosol, inoculated to 5 mL of a selective medium for screening auxotrophic yeast, incubated at 30-32° C. and 200 rpm for 24 h, further inoculated in 50 mL of a selective medium for screening auxotrophic yeast at an initial OD600 of 0.2, incubated at 30° C. and 200 rpm for 12 h, and further inoculated in 100 mL of a selective medium for screening auxotrophic yeast at an initial OD600 of 0.2. Glucose, fructose, sucrose, glucose and tyrosine were respectively added for 72-h fermentation experiment. The tyrosol concentration in the fermentation broth was detected by the HPLC method reported in the literature (Satoh, Tajima et al. 2012). The amounts of tyrosol produced by culturing in the presence of various carbon sources are shown in Table 4.

TABLE-US-00004 TABLE 4 Amounts of tyrosol produced by 72-h fermentation in the presence of various carbon sources 2% 2% glucose + 2% 2% glucose 1% tyrosine fructose sucrose Strain (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) PcAAS-ADH-ΔPDC1-TyrA 1103.16 1326.18 964.72 724.37 PcAAS-ADH 941.42 846.96 1121.08 1038.11 BY4741 34.33 35.65 38.32 36.54

Example 7

[0088] Fermentation with Microorganisms Synthesizing Hydroxytyrosol, with Saccharomyces cerevisiae as an Example:

[0089] Single clones were picked up from a plate containing the PcAAS-ADH-ΔPDC1-TyrA-HpaBC strain producing hydroxytyrosol, inoculated to 5 mL of a selective medium for screening auxotrophic yeast, incubated at 30-32° C. and 200 rpm for 24 h, further inoculated in 50 mL of a selective medium for screening auxotrophic yeast at an initial OD600 of 0.2, incubated at 30° C. and 200 rpm for 12 h, and further inoculated in 100 mL of a selective medium for screening auxotrophic yeast at an initial OD600 of 0.2 for 72-h fermentation experiment. The hydroxytyrosol concentration in the fermentation broth was detected by the HPLC method reported in the literature (Satoh, Tajima et al. 2012). 978 mg/L of hydroxytyrosol was obtained after 72-h fermentation.

[0090] The foregoing descriptions are merely preferred embodiments of this application, but are not intended to limit this application. A person of ordinary skill in the art may make various alterations and variations to this application. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of this application shall fall within the protection scope of this application.