Saccharomyces cerevisiae recombinant bacterium for valene biosynthesis and application of saccharomyces cerevisiae recombinant bacterium

Abstract

The invention discloses a saccharomyces cerevisiae recombinant bacterium for valene biosynthesis and application thereof. The recombinant bacterium is constructed by the following steps: jointly integrating a valene synthase expression cassette and an LEU2 nutrition tag at a YMR038C ARS site of a saccharomyces cerevisiae chassis bacterium, a YOR317W ARS site of the saccharomyces cerevisiae chassis bacterium or a YPR028W ARS site of the saccharomyces cerevisiae chassis bacterium; sequentially obtaining a saccharomyces cerevisiae recombinant bacterium 1, a saccharomyces cerevisiae recombinant bacterium 2 or a saccharomyces cerevisiae recombinant bacterium 3 for valene biosynthesis; experiments prove that the recombinant bacteria are obtained by integrating a valene synthase expression cassette on three high-activity ARS sites of YMR038C, YOR317W and YPR028W on a saccharomyces cerevisiae chassis bacterial genome for the first time, and fermentation verifies that the production of the valene is realized by the fermentation of the recombinant saccharomyces cerevisiae.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosynthesis, and specifically relates to a recombinant Saccharomyces cerevisiae strain for valencene biosynthesis and its application. Background Art

[0002] Valencene is a sesquiterpene of natural origin (CAS No.: 4630-07-3), which has potential effects of anti-inflammatory, antibacterial, and neuroprotection, and shows great prospects in the applications in industries such as food, cosmetics, pharmaceuticals, chemistry, and agriculture. At present, the production of valencene is mainly natural extraction and chemical synthesis, but the low efficiency, high consumption, and high pollution of these two methods have become bottleneck factors restricting the application of valencene. The method of microbial synthesis of valencene has the advantages of low pollution and low cost, and has become the preferred method for the production of natural compounds including valencene.

[0003] At present, valencene has been successfully synthesized in microbial chassis such as Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, cyanobacteria, and Corynebacterium glutamicum. Methods such as gene screening, protein engineering, and biosynthetic pathway optimization are used to transform the microbial chassis to improve the yield. However, in the currently disclosed technologies, the fermentation yield of valencene is relatively low.

[0004] For the existing Saccharomyces cerevisiae to synthesize valencene, commonly used genomic integration sites such as URA3 and LEU2 are generally adopted, and other potential sites with higher ARSs activity are not selected. Therefore, in terms of the dimension of genomic integration sites, there is a certain room for improvement in the yield of valencene biosynthesis. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a recombinant Saccharomyces cerevisiae strain for valencene biosynthesis.

[0006] The second purpose of the present invention is to provide a construction method of a recombinant Saccharomyces cerevisiae strain for valencene biosynthesis.

[0007] The third purpose of the present invention is to provide an application of a recombinant Saccharomyces cerevisiae strain for valencene biosynthesis in the fermentation preparation of valencene.

[0008] The technical solution of the present invention is outlined as follows:

[0009] A construction method of a recombinant Saccharomyces cerevisiae strain for valencene biosynthesis, comprising the following steps:

[0010] Integrate the valencene synthase expression cassette and the LEU2 nutritional label into the YMR038C ARS locus of the Saccharomyces cerevisiae chassis strain, the YOR317W ARS locus of the Saccharomyces cerevisiae chassis strain, or the YPR028W ARS locus of the Saccharomyces cerevisiae chassis strain to successively obtain Saccharomyces cerevisiae recombinant strain 1, recombinant strain 2, or recombinant strain 3 for valencene biosynthesis;

[0011] The nucleotide sequence of the YMR038C ARS locus is shown in SEQ ID NO.3;

[0012] The nucleotide sequence of the YOR317W ARS locus is shown in SEQ ID NO.4;

[0013] The nucleotide sequence of the YPR028W ARS locus is shown in SEQ ID NO.5.

[0014] Preferably, the valencene synthase expression cassette consists of a GPD promoter, a valencene synthase gene optimized and truncated with Saccharomyces cerevisiae codons, and a GPD terminator; the nucleotide sequence of the valencene synthase gene optimized and truncated with Saccharomyces cerevisiae codons is shown in SEQ ID NO.2

[0015] Saccharomyces cerevisiae recombinant strains for valencene biosynthesis constructed by the above construction method.

[0016] Application of the above Saccharomyces cerevisiae recombinant strains for valencene biosynthesis in fermenting and preparing valencene.

[0017] Advantages of the present invention:

[0018] Experiments have proved that the present invention first uses the valencene synthase expression cassette to integrate into three highly active ARS loci, namely YMR038C, YOR317W, and YPR028W, on the genome of the Saccharomyces cerevisiae chassis strain to obtain recombinant strains, and fermentation has verified that recombinant Saccharomyces cerevisiae can ferment to produce valencene. Description of the Drawings

[0019] Figure 1 For the gene integration sites and valencene fermentation yields of 16 Saccharomyces cerevisiae recombinant strains. Detailed Embodiments

[0020] The following combines specific embodiments to further illustrate the present invention.

[0021] Example 1

[0022] The yeast strain chassis used in the present invention is Saccharomyces cerevisiae SyBE_Sc01130007 with enhanced metabolic flux of the MVA pathway, which is disclosed in the invention patent "A Gene, Recombinant Saccharomyces cerevisiae Strain and Its Construction Method and Application" applied by Tianjin University, with the patent application number: 201710086073, CN106754993B, and has been authorized.

[0023] The ERG5 gene was further knocked out from the SyBE_Sc01130007 strain, and the obtained strain was named SyBE_Sc0125XJ01, which is the Saccharomyces cerevisiae chassis strain used in the present invention.

[0024] Example 2

[0025] A method for constructing a recombinant Saccharomyces cerevisiae strain for valencene biosynthesis, comprising the following steps:

[0026] Integrate the valencene synthase expression cassette and the LEU2 nutritional label into the YMR038C ARS locus of the Saccharomyces cerevisiae chassis strain (SyBE_Sc0125XJ01), the YOR317W ARS locus of the Saccharomyces cerevisiae chassis strain (SyBE_Sc0125XJ01), or the YPR028W ARS locus of the Saccharomyces cerevisiae chassis strain (SyBE_Sc0125XJ01) in sequence to obtain recombinant Saccharomyces cerevisiae strain 1, recombinant strain 2, or recombinant strain 3 for valencene biosynthesis;

[0027] Saccharomyces cerevisiae

[0028] Valencene synthase EgVS, derived from Eryngium glaciale, the truncated 16 - amino - acid EgVS amino acid sequence is shown in SEQ ID NO.1.

[0029] The nucleotide sequence of the YMR038C ARS locus is shown in SEQ ID NO.3;

[0030] The nucleotide sequence of the YOR317W ARS locus is shown in SEQ ID NO.4;

[0031] The nucleotide sequence of the YPR028W ARS locus is shown in SEQ ID NO.5.

[0032] The valencene synthase expression cassette consists of a GPD promoter (well - known), the valencene synthase gene optimized with Saccharomyces cerevisiae codons and truncated, denoted as t16 - EgVS (SEQ ID NO.2), and a GPD terminator (well - known).

[0033] The codon-optimized and truncated valencene synthase gene of Saccharomyces cerevisiae, wherein the truncation is achieved by deleting the first 45 DNA sequences (excluding the start codon ATG) of the EgVS gene from the valencene synthase gene.

[0034] Example 3

[0035] In the present invention, a homologous recombination scheme is adopted for the genomic integration of the valencene synthase expression cassette. The selected genomic integration sites are all ARSs sites with strong activity, that is, after integrating the red fluorescent protein gene RFP at the adjacent sites of these ARSs, the tested relative fluorescence intensity is 8 or above. The selected genomic integration sites are: SWH1 (YAR042W), ATP1 (YBL099W), ADP1 (YCR011C), ADA2 (YDR448W), YER137C, RIM15 (YFL033C), CHO2 (YGR157W), STB5 (YHR178W), EST3 (YIL009C-A), VPS35 (YJL154C), CNB1 (YKL190W), SAM1 (YLR180W), CCS1 (YMR038C), SKP2 (YNL311C), FAA1 (YOR317W), YOP1

[0036] (YPR028W).

[0037] Using YMR038C-U-F (SEQ ID NO.10) and YMR038C-U-R (SEQ ID NO.11) as primers, with the genome of the Saccharomyces cerevisiae strain SyBE_Sc0125XJ01 as the template, PCR the left homologous arm sequence-1 using KOD ONE enzyme;

[0038] Using YMR038C-D-F (SEQ ID NO.6) and YMR038C-D-R (SEQ ID NO.7) as primers, with the genome of the Saccharomyces cerevisiae strain SyBE_Sc0125XJ01 as the template, PCR the right homologous arm sequence-1 using KOD ONE enzyme;

[0039] Using PGPD-F (SEQ ID NO.26) and GPDt-R (SEQ ID NO.24) as primers,

[0040] Using the valencene synthase expression cassette as the template, PCR the valencene synthase expression cassette using KOD ONE enzyme;

[0041] Using PLEU2-F (SEQ ID NO.27) and LEU2-R (SEQ ID NO.25) as primers, with the pRS415 plasmid (commercial) as the template, PCR the LEU2 nutritional label using KOD ONE enzyme;

[0042] There is a homologous sequence of more than 20 bp between the above 4 fragments, which facilitates subsequent ligation.

[0043] Use the OE PCR method to combine the four DNA fragments in pairs. Specifically,

[0044] Using YMR038C-U-F (SEQ ID NO.10) and GPDt-R (SEQ ID NO.24) as primers,

[0045] Using the left homologous arm-1 sequence and the valencene synthase expression cassette as templates, and using KOD ONE enzyme to combine the two to obtain combined fragment 1;

[0046] Using PLEU2-F (SEQ ID NO.27) and YMR038C-D-R (SEQ ID NO.7) as primers,

[0047] Using the LEU2 nutritional label and the right homologous arm-1 sequence as templates, and using KOD ONE enzyme to combine the two to obtain combined fragment 2.

[0048] The two (combined fragment 1 and combined fragment 2) are DNA fragments for yeast transformation and integration into the genome.

[0049] Using the two DNA fragments (combined fragment 1 and combined fragment 2) at each locus as transformation fragments. Using the SyBE_Sc0125XJ01 strain as the chassis, perform yeast transformation and spread it on a solid SC-Leu plate. Subsequently, perform yeast colony PCR. Using the system after boiling a single colony as the template, and using YMR038C-F-screen (SEQ ID NO.8) and PGPD-R-screen (SEQ ID NO.29), YMR038C-R-screen (SEQ ID NO.9) and LEU2-F-screen (SEQ ID NO.28) as two pairs of primers, screen to obtain yeast single colony 1 of positive clones.

[0050] Inoculate a single yeast colony 1 into 2 ml of YPD liquid medium and culture it at 30 °C with 250 rpm for 16 hours. Take 1 ml of the bacterial liquid to extract the genome. Using this as a template, perform PCR amplification of the DNA sequence with YMR038C-F-screen (SEQ ID NO.8) and YMR038C-R-screen (SEQ ID NO.9) as primers, and send the sample for Sanger sequencing. The sequencing primers used are: PGPD-F-sanger (SEQ ID NO.33), PGPD-R-sanger (SEQ ID NO.34), GPDt-F-sanger (SEQ ID NO.32), DQ-54-F-sanger-1 (SEQ ID NO.30), and DQ-54-F-sanger-2 (SEQ ID NO.31). Select the single colony with correct sequencing results as the successfully constructed valencene-producing yeast strain, abbreviated as recombinant bacterium 1 (i.e., Figure 1 YMR038C in

[0051] Example 4

[0052] Using YOR317W-U-F (SEQ ID NO.16) and YOR317W-U-R (SEQ ID NO.17) as primers, and the genome of Saccharomyces cerevisiae strain SyBE_Sc0125XJ01 as a template, use KOD ONE enzyme to perform PCR amplification of the left homologous arm-2 sequence;

[0053] Using YOR317W-D-F (SEQ ID NO.12) and YOR317W-D-R (SEQ ID NO.13) as primers, and the genome of Saccharomyces cerevisiae strain SyBE_Sc0125XJ01 as a template, use KOD ONE enzyme to perform PCR amplification of the right homologous arm-2 sequence;

[0054] Using PGPD-F (SEQ ID NO.26) and GPDt-R (SEQ ID NO.24) as primers,

[0055] Using the valencene synthase expression cassette as a template, use KOD ONE enzyme to perform PCR amplification of the valencene synthase expression cassette;

[0056] Using PLEU2-F (SEQ ID NO.27) and LEU2-R (SEQ ID NO.25) as primers, and the pRS415 plasmid as a template, use KOD ONE enzyme to perform PCR amplification of the LEU2 nutritional label,

[0057] There is more than 20 bp of homologous sequence between the above 4 fragments, which is convenient for subsequent ligation.

[0058] The four DNA fragments were combined in pairs using the OE PCR method. Specifically, using YOR317W-U-F (SEQ ID NO.16) and GPDt-R (SEQ ID NO.24) as primers, and the left homologous arm-2 sequence and the valencene synthase expression cassette as templates, the two were combined using KOD ONE enzyme to obtain combined fragment 3; using PLEU2-F (SEQ ID NO.27) and YOR317W-D-R (SEQ ID NO.13) as primers, and the LEU2 nutritional label and the right homologous arm-2 sequence as templates, the two were combined using KOD ONE enzyme to obtain combined fragment 4, and two DNA fragments (combined fragment 3 and combined fragment 4) for yeast transformation and integration into the genome were obtained.

[0059] Using the two DNA fragments at each locus as transformation fragments and the SyBE_Sc0125XJ01 strain as the chassis, yeast transformation was carried out and plated on a solid SC-Leu plate. Subsequently, yeast colony PCR was performed, using the system after boiling a single colony as the template, and YOR317W-F-screen (SEQ ID NO.14) and PGPD-R-screen (SEQ ID NO.29), YOR317W-R-screen (SEQ ID NO.15) and LEU2-F-screen (SEQ ID NO.28) as two pairs of primers, and yeast single colonies 2 of positive clones were screened.

[0060] Yeast single colony 2 was inoculated into 2 ml of YPD liquid medium and cultured at 30 °C and 250 rpm for 16 hours. 1 ml of the bacterial solution was taken to extract the genome. Using this as the template, PCR amplification of the DNA sequence was carried out using YOR317W-F-screen (SEQ ID NO.14) and YOR317W-R-screen (SEQ ID NO.15) as primers, and the sample was sent for sanger sequencing. The sequencing primers used were: PGPD-F-sanger (SEQ ID NO.33), PGPD-R-sanger (SEQ ID NO.34), GPDt-F-sanger (SEQ ID NO.32), DQ-54-F-sanger-1 (SEQ ID NO.30) and DQ-54-F-sanger-2 (SEQ ID NO.31). The single colony with the correct sequencing result was selected as the successfully constructed valencene-producing yeast strain, abbreviated as recombinant strain 2 (i.e., Figure 1 YOR317W in

[0061] Example 5

[0062] Using YPR028W-U-F (SEQ ID NO.22) and YPR028W-U-R (SEQ ID NO.23) as primers, with the genome of Saccharomyces cerevisiae strain SyBE_Sc0125XJ01 as the template, PCR the left homologous arm-3 sequence using KOD ONE enzyme; using YPR028W-D-F (SEQ ID NO.18) and YPR028W-D-R (SEQ ID NO.19) as primers, with the genome of Saccharomyces cerevisiae strain SyBE_Sc0125XJ01 as the template, PCR the right homologous arm-3 sequence using KOD ONE enzyme;

[0063] Using PGPD-F (SEQ ID NO.26) and GPDt-R (SEQ ID NO.24) as primers, with the valencene synthase expression cassette as the template, PCR the valencene synthase expression cassette using KOD ONE enzyme;

[0064] Using PLEU2-F (SEQ ID NO.27) and LEU2-R (SEQ ID NO.25) as primers, with the pRS415 plasmid as the template, PCR the LEU2 nutritional label using KOD ONE enzyme,

[0065] There are homologous sequences of more than 20 bp between the above four fragments, which facilitates subsequent ligation.

[0066] Using the OE PCR method to combine the four DNA fragments in pairs. Specifically, using YPR028W-U-F (SEQ ID NO.22) and GPDt-R (SEQ ID NO.24) as primers, with the left homologous arm-3 sequence and the valencene synthase expression cassette as the template, use KOD ONE enzyme to combine them to obtain combined fragment 5; using PLEU2-F (SEQ ID NO.27) and YPR028W-D-R (SEQ ID NO.19) as primers, with the LEU2 nutritional label and the right homologous arm-3 sequence as the template, use KOD ONE enzyme to combine them to obtain combined fragment 6, and obtain two DNA fragments (combined fragment 5 and combined fragment 6) for yeast transformation and integration into the genome.

[0067] Using two DNA fragments at each locus as transformation fragments, and strain SyBE_Sc0125XJ01 as the chassis, yeast transformation was carried out and spread on a solid SC-Leu plate. Subsequently, yeast colony PCR was performed, using the system after boiling a single colony as the template, and YPR028W-F-screen (SEQ ID NO.20) and PGPD-R-screen (SEQ ID NO.29), YPR028W-R-screen (SEQ ID NO.21) and LEU2-F-screen (SEQ ID NO.28) as two pairs of primers, and 3 yeast single colonies of positive clones were screened out.

[0068] The yeast single colonies were inoculated into 2 ml of YPD liquid medium and cultured at 30 °C and 250 rpm for 16 hours. 1 ml of the bacterial solution was taken to extract the genome. Using this as the template, the DNA sequence was amplified by PCR with YPR028W-F-screen (SEQ ID NO.20) and YPR028W-R-screen (SEQ ID NO.21) as primers, and the samples were sent for Sanger sequencing. The sequencing primers used were: PGPD-F-sanger (SEQ ID NO.33), PGPD-R-sanger (SEQ ID NO.34), GPDt-F-sanger (SEQ ID NO.32), DQ-54-F-sanger-1 (SEQ ID NO.30) and DQ-54-F-sanger-2 (SEQ ID NO.31). The single colonies with correct sequencing results were selected as the successfully constructed valencene-producing yeast strains, abbreviated as recombinant strain 3 (i.e., Figure 1 YPR028W in

[0069] Two-phase fermentation: After the construction of Saccharomyces cerevisiae, it was first streaked and purified on a solid SC-Leu plate, and then a single colony was picked and cultured in 3 ml of SC-ura liquid medium for 24 hours (primary seed solution). Then, it was transferred and inoculated into 3 ml of SC-ura liquid medium at an initial OD 600 = 0.2 and cultured for about 15 hours to obtain the secondary seed solution, with an initial OD 600Inoculate 0.2 into a 250 ml shake flask containing 25 ml of SC-Leu liquid medium. The medium also contains 10 g / L of galactose for induction of expression, and 5 ml of the organic phase isopropyl myristate (IPM) is covered for extraction of the product valencene. There are three parallel samples in each group. After 72 hours of fermentation, centrifuge the fermentation broth at 12,000 rpm for 10 min. Take 1 ml of the upper organic phase and put it into a 1.5 ml centrifuge tube. Add an appropriate amount of anhydrous sodium sulfate for water removal. After standing for 20 min, take the upper organic phase, dilute it five times, filter it with an organic phase filter membrane and then load the sample. The titer of valencene is determined by gas chromatography-tandem mass spectrometry (GC-MS).

[0070] The fermentation results of 16 strains are as Figure 1 shown.

Claims

1. A method for constructing a recombinant Saccharomyces cerevisiae strain for valencene biosynthesis, characterized in that It includes the following steps: Integrate the valencene synthase expression cassette and the LEU2 nutritional label into the YMR038C ARS locus of the Saccharomyces cerevisiae chassis bacterium, the YOR317W ARS locus of the Saccharomyces cerevisiae chassis bacterium, or the YPR028W ARS locus of the Saccharomyces cerevisiae chassis bacterium, successively to obtain the Saccharomyces cerevisiae recombinant bacterium 1, recombinant bacterium 2, or recombinant bacterium 3 for valencene biosynthesis; The nucleotide sequence of the YMR038C ARS locus is as shown in SEQ ID NO.3; The nucleotide sequence of the YOR317W ARS locus is as shown in SEQ ID NO.4; The nucleotide sequence of the YPR028W ARS locus is as shown in SEQ ID NO.

5.

2. The method according to claim 1, characterized in that The valencene synthase expression cassette consists of a GPD promoter, a valencene synthase gene optimized and truncated with Saccharomyces cerevisiae codons, and a GPD terminator; the nucleotide sequence of the valencene synthase gene optimized and truncated with Saccharomyces cerevisiae codons is as shown in SEQ ID NO.

2.

6. The Saccharomyces cerevisiae recombinant bacterium for valencene biosynthesis constructed by the construction method according to claim 1 or 2.

7. Use of the Saccharomyces cerevisiae recombinant bacterium for valencene biosynthesis according to claim 3 for fermenting and preparing valencene.

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