CN107916274B - Method for increasing production of S-adenosylmethionine by modulating carbon metabolic pathway genes - Google Patents

Abstract

The present invention relates to methods for increasing the production of S-adenosylmethionine by modulating carbon metabolic pathway genes. The invention aims to search a modification target point influencing the synthesis of a target product S-adenosylmethionine (SAM) from the whole genome range, and firstly discloses that the S-adenosylmethionine yield of an S-adenosylmethionine production strain can be obviously improved by transferring the coding gene of zwf1 or sol3 into the S-adenosylmethionine production strain.

Description

Methods for increasing S-adenosylmethionine production by modulating carbon metabolism pathway genes

technical field

The invention belongs to the field of bioengineering, and more particularly, the invention relates to a method for improving the production of S-adenosylmethionine by regulating carbon metabolism pathway genes.

Background technique

SAM is ubiquitous in animals, plants and microorganisms. It is an important intermediate metabolite in organisms and participates in various biochemical reactions in the body. It was discovered by Italian scientists Cantoni et al. SAM has a very high medical value and has a significant effect on the treatment of depression, liver disease, arthritis and other diseases. Therefore, how to efficiently and cheaply produce SAM is the key to its large-scale production. At present, the method of metabolic engineering is mainly used to obtain SAM high-producing strains to improve SAM production.

So far, there are four main ways to use metabolic engineering to change the cell metabolism of yeast, so as to obtain SAM high-producing bacteria.

One is to overexpress S-adenosylmethionine synthase (MAT) to increase the activity of SAM synthase and promote the synthesis of SAM. There are two kinds of SAM synthases in the organism, the presence of methionine or SAM can prevent the normal expression of sam1 gene, but sam2 gene can encode a highly active SAM synthase under this condition, and make its expression level A significant increase. Therefore, Li et al. (Li D Y, Yu J, Tian L, et al. Production of SAM by recombinant Pichia pastoris [J]. Journal of Biotechnology. 2002, 18: 295-299) transformed the sam2 gene into Pichia pastoris GS115, The expression of sam2 gene was regulated by AOX1 promoter, which increased the SAM synthase activity by 37 times, and the SAM yield reached 1.58g/L. Since the AOX1 promoter is a methanol-inducible promoter, the gene expression can be induced only in the presence of methanol. The presence of glycerol will inhibit the induction of the AOX1 promoter, but given that methanol is toxic to cells, during the induction process Only add in small amounts. Therefore, Yu et al. (Yu Z L, Wu X J, Li D Y, et al. Enhancement of the production of SAM by overexpression of SAM synthetase in Pichia pastoris [J]. Acta Biochimica et Biophysica Sinica. 2003, 35: 127-132.) tried to use Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter regulates the expression of sam2 gene in Pichia pastoris, and the SAM yield reaches 2.49g/L. Hu et al. (Hu H, Qian J C, Chu J, et al. DNA shuffling of methionine adenosyltransferase geneleads to improved S-adenosyl-L-methionine production in Pichia pastoris [J]. Journal of Biotechnology. 2009, 141:97-103. ) The SAM synthases derived from Streptomyces, Saccharomyces cerevisiae and Escherichia coli were evolved in vitro by DNA Shuffling technology, and the SAM synthase with high activity was screened out and transferred into Pichia pastoris GS115 to obtain recombinant strain DS16 , the yield of SAM on the 500L fermenter reached 6.14g/L.

The second is to knock out the cys4 gene (encoding cystathionine β-synthase), inhibit the activity of cystathionine β-synthase, and effectively reduce the intracellular conversion of SAM to cysteine. He et al. (He J Y, Deng J J, Zheng Y H, et al. Asynergistic effect on the production of S-adenosyl-L-methionine in Pichiapastoris by knocking in of S-adenosyl-L-methionine synthase and knocking out of cystathionine-beta synthase[ J]. Journal of Biotechnology. 2006, 126: 519-527.) The cys4 gene was knocked out by gene knockout, and the result was that the accumulation of SAM was more than doubled, but the SAM catabolism pathway was completely blocked, and the recombinant bacteria would To become auxotrophic, exogenous addition of reduced glutathione (GSH) is required in the medium, resulting in greatly increased fermentation costs. Therefore, our laboratory Qin (Qin XL, Qian J C, Yao GF, et al. GAP promoter library for fine-tuning of gene expression in Pichia pastoris[J]. Applied and Environmental Microbiology. 2011, 77: 3600-3608.) used GAP to start The sub-library weakens the cys4 gene expression, making it no longer auxotrophic and can accumulate a large amount of SAM in the cells, and obtain a SAM high-producing strain G12, which can reach 133.2mg/g DCW by shaking flask culture, and recombined in a 5L tank The SAM yield of bacteria G12 reached 146.8 mg/g DCW.

The third is to change the regulation of methyltetrahydrofolate reductase (MTHFR) on SAM, relieve its feedback inhibition on SAM, and promote the synthesis of SAM. In S. cerevisiae, MTHFR has two isozymes, encoded by the MET12 and MET13 genes, respectively. Therefore, Sanja et al. (Roje S, Chan S Y, Kaplan F, et al. Metabolic engineering in yeastdemonstrates that S-adenosylmethionine controls flux through themethylenetetrahydrofolate reductase) reaction in vivo [J]. Journal of Biological Chemistry. 2002, 277:36904.) constructed a chimeric MTHFR (Chimera-1) consisting of the N-terminal catalytic site of yeast Met13p and the C-terminal regulatory site of Arabidopsisthaliana MTHFR (AMTHFR-1). ), the feedback inhibition of SAM on MTHFR was relieved, and the SAM yield of the genetically engineered yeast SCY4 was 140 times higher than that of the wild-type strain.

The fourth is to knock out the ERG6 gene encoding Δ-24-sterol-C-transmethylase to block the sterol synthesis pathway and reduce the consumption of SAM. Shobayashi et al. (Shobayashi M, Mukai N, Iwashita K, et al. A new method forisolation of S-adenosy-L-methionine (SAM)-accumulating yeast[J]. Applied Microbiology and Biotechnology. 2006, 69:704-710.) The ERG6 gene from Saccharomyces cerevisiae K-9 and X2180-1A was knocked out, and mutants from K-9 and X2180-1A were obtained, respectively. The fermentation results showed that the SAM production from K-9 and X2180-1A mutants They were 1.7-3 times and 4.2-5.5 times higher than the starting strains, respectively.

The above methods only modify the local pathways of SAM synthesis and degradation, which have certain limitations. Those skilled in the art also need to use the methods of systems biology to analyze the physiological characteristics of cells, and to search for modified targets that affect the synthesis of target products from the whole genome.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for increasing the production of S-adenosylmethionine by regulating carbon metabolism pathway genes.

In a first aspect of the present invention, there is provided a method for producing S-adenosylmethionine, the method comprising:

(1) Introduction of exogenous Glucose-6-phosphate dehydrogenase (Glucose-6-phosphate dehydrogenase; zwf1) expression cassette or 6-phosphogluconolactonase ( 6-phosphogluconolactonase; sol3) expression cassette;

(2) The strain prepared in step (1) is cultured to produce S-adenosylmethionine.

In a preferred example, the expression cassette for glucose-6-phosphate dehydrogenase includes the following operably linked elements: AOX1 promoter, a gene encoding glucose-6-phosphate dehydrogenase, and a transcription termination sequence.

In another preferred embodiment, the 6-phosphogluconolactonase expression cassette includes the following operably linked elements: AOX1 promoter, a gene encoding 6-phosphogluconolactonase, and a transcription termination sequence.

In another preferred embodiment, the S-adenosylmethionine-producing strain is (a) a strain with SAM synthase gene ds16 inserted and β-cystathionine synthase gene weakened (eg G12) or (b) on the basis of (a) strains, the strains of the selectable markers are excluded, such as G12', which is based on G12 with the MazF-ZeoR marker recovery unit to achieve the recovery of the selectable markers, so that the new recombinant bacteria G12 'Without Zeocin resistance, the reuse of the ZeoR marker in Pichia pastoris was achieved.

In another preferred example, in step (2) of the method, the method for culturing includes: inducing culture at 30±2° C. and 220±50 rpm; after adding methanol to the final concentration of 1±0.2% for the first time, every other Methanol was added for 12±2h to a final concentration of 1±0.2% by volume; L-Met was added every 24±4h, and a total of 96±12h was induced.

In another aspect of the present invention, an improved S-adenosylmethionine production strain is provided, the genome of the strain has introduced an exogenous glucose-6-phosphate dehydrogenase expression cassette or glucose-6-phosphate Acid lactonase expression cassette.

In a preferred example, the 6-phosphate glucose dehydrogenase expression cassette includes the following operably linked elements: AOX1 promoter, a gene encoding 6-phosphate glucose dehydrogenase, and a transcription termination sequence.

In another preferred embodiment, the 6-phosphogluconolactonase expression cassette includes the following operably linked elements: AOX1 promoter, a gene encoding 6-phosphogluconolactonase, and a transcription termination sequence.

In another preferred embodiment, the improved S-adenosylmethionine producing strain is: (a) a strain with the SAM synthase gene ds16 inserted and the β-cystathionine synthase gene weakened; or (b) On the basis of (a) strains, strains with selectable markers were excluded.

In another aspect of the present invention, there is provided the use of glucose-6-phosphate dehydrogenase for promoting the production of S-adenosylmethionine by an S-adenosylmethionine producing strain.

In another aspect of the present invention, there is provided the use of 6-phosphogluconolactonase for promoting the production of S-adenosylmethionine by an S-adenosylmethionine producing strain.

In another aspect of the present invention, there is provided a kit for producing S-adenosylmethionine, the kit comprising the improved S-adenosylmethionine producing strain.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. Schematic diagram of the construction process of plasmids pAOX-zwf1 and pAOX-sol3.

Figure 2. Schematic diagram of the construction process of recombinant bacteria G12'/AOX-zwf1 and G12'/AOX-sol3. The genomic DNA extracted from the transformants was verified by PCR and sent for sequencing verification.

Figure 3. The effect of enhancing the zwf1 and sol3 genes on the growth of recombinant bacteria.

Figure 4. The effect of enhancing zwf1 and sol3 genes on SAM synthesis in recombinant bacteria.

Figure 5. Schematic diagram of the construction process of plasmid pCmG(R-A-D).

Figure 6. Schematic diagram of the construction process of plasmid pAOX-gln.

Figure 7. Schematic diagram of the construction process of plasmid pAOX(SSN).

Figure 8. Schematic diagram of the construction of strain G12'.

Detailed ways

The present invention is devoted to searching for the transformation target affecting the synthesis of the target product S-adenosylmethionine (SAM) from the whole genome, and it is revealed for the first time that the encoding gene of zwf1 or sol3 is transferred into the S-adenosylmethionine producing strain , can significantly improve the S-adenosylmethionine production of S-adenosylmethionine producing strains.

As used herein, "foreign" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest does not normally occur in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

As used herein, the "promoter" refers to a nucleic acid sequence, usually present upstream (5' end) of the coding sequence of a gene of interest, capable of directing the transcription of the nucleic acid sequence into mRNA. Typically, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for the proper initiation of transcription. As used herein, the promoter or promoter region includes active variants of the promoter, which may be naturally occurring allelic variants or non-naturally occurring variants. The variants include substitution variants, deletion variants and insertion variants.

As used herein, the "expression cassette" refers to a gene expression system that contains all necessary elements for expressing a polypeptide of interest, typically it includes the following elements: a promoter, a gene sequence encoding the polypeptide, a terminator; Alternatives include signal peptide coding sequences and the like. These elements are operatively connected.

As used herein, "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, the promoter region is placed at a specific location relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, and thus, the promoter region is "operably linked" to the nucleic acid sequence.

NCBI has published the zwf1 polypeptide sequence or the zwf1 gene sequence, therefore, those in the art can easily obtain and prepare the polypeptide or gene. As a preferred mode of the present invention, the zwf1 has the amino acid sequence shown in SEQ ID NO:2, and the zwf1 has the nucleotide sequence shown in SEQ ID NO:1.

The sol3 polypeptide sequence or the sol3 gene sequence has been published by NCBI, therefore, the polypeptide or gene can be easily obtained and prepared by those skilled in the art. As a preferred mode of the present invention, the zwf1 has the amino acid sequence shown in SEQ ID NO:4, and the zwf1 has the nucleotide sequence shown in SEQ ID NO:3.

In the present invention, the term "zwf1 polypeptide" refers to the polypeptide of the sequence of SEQ ID NO: 2 having the function of increasing the production of SAM; the term also includes the variant form of the sequence of SEQ ID NO: 2 having the function of increasing the production of SAM. The term "sol3 polypeptide" refers to a polypeptide of the sequence of SEQ ID NO: 4 having the function of increasing SAM production; the term also includes variant forms of the sequence of SEQ ID NO: 4 having the function of increasing the production of SAM. These variants include (but are not limited to): several (eg 1-50, more preferably 1-30, more preferably 1-20, still more preferably 1-10, 1-5 or 1-3) amino acid deletion, insertion and/or substitution, and addition or deletion of one or several (such as 1-50, more preferably 1-30, preferably 1-50, preferably 1-30) at the C-terminal and/or N-terminal 1-20, more preferably 1-10, such as 1-5 or 1-3) amino acids. Said variant forms all have the function of increasing the yield of SAM.

Variant forms of polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, those that can hybridize to the DNA of zwf1 polypeptide and/or sol3 polypeptide under high or low stringency conditions Polypeptides encoded by DNA.

The present invention also relates to polynucleotide variants of zwf1 or sol3, which encode the same polypeptide as the amino acid sequence encoded by the wild-type gene. Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides that does not substantially alter the function of the encoded polypeptide .

The full-length nucleotide sequence of zwf1 or sol3 of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercial cDNA libraries or cDNAs prepared by conventional methods known to those skilled in the art can be used. The library is used as a template to amplify the relevant sequences.

In order to optimize the production of S-adenosylmethionine by S-adenosylmethionine producing strains, the present inventors have conducted extensive research, from the aspect of Pichia pastoris carbon metabolism, to find metabolic targets, and combined with Recombinant bacteria were constructed by genetic engineering technology to investigate the effect of SAM synthesis. The synthesis of SAM requires ATP to provide precursors, as well as energy and reducing power to promote bacterial growth and maintenance. In the Pichia pastoris carbon metabolism network, the pentose phosphate pathway (PPP) is the main source of reducing power such as NADPH. The inventors found that strengthening this PPP pathway may improve the level of energy and reducing power in cells, which is beneficial to bacteria. body growth and SAM synthesis. On this basis, the inventors found zwf1 or sol3 genes suitable for improvement, and constructed corresponding constructs.

Therefore, the present invention provides a construct comprising: an expression cassette of zwf1 or sol3. The expression cassette has all the elements required for gene expression (including promoter, coding DNA, terminator, etc.), so that the corresponding zwf1 or sol3 polypeptide can be completely expressed.

Some inducible or constitutive promoters known in the art are suitable for use in the present invention for the construction of expression cassettes. As a preferred mode of the present invention, the promoter is the methanol-inducible promoter AOX1 promoter. Some strong promoters are preferred when used to enhance the expression of a gene.

Typically, the construct is located on an expression vector. Accordingly, the present invention also includes a vector containing the construct. The expression vector usually also contains an origin of replication and/or marker gene and the like. Methods well known to those skilled in the art can be used to construct the desired expression vectors of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator.

In order to achieve gene-directed knockout and gene-directed recombination on the genome, the expression vector can also be provided with suitable homology arms; the setting of homology arms is understood by those skilled in the art. It should be understood that any expression vector can be selected as long as the transformation of the S-adenosylmethionine-producing strain of the present invention can be achieved. Furthermore, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

Vectors comprising the appropriate polynucleotide sequences described above, together with appropriate promoter or control sequences, can be used to transform appropriate hosts. In the method of the present invention, the host is an S-adenosylmethionine producing strain. As a preferred mode of the present invention, the S-adenosylmethionine-producing strain is a yeast strain, preferably a Pichia strain, including various modified Pichia strains; most preferably, S-adenosylmethionine The glycoside methionine-producing strain is the G12' strain, which is based on DS16 (Hu H, Qian J C, Chu J, et al. DNA shuffling of methionine adenosyltransferasegene leads to improved S-adenosyl-L-methionine production in Pichia pastoris[ J]. Journal of Biotechnology. 2009, 141: 97-103) as the starting strain, a recombinant Pichia strain whose G12 promoter regulates the expression of CBS on the chromosome. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually based on conventional conditions such as those described in J. Sambrook et al., "Guide to Molecular Cloning", 3rd edition, Science Press, 2002, or according to the conditions described in the manufacture. conditions recommended by the manufacturer.

I. Materials and Methods

1. Plasmids, strains and primers

In the following examples of the present invention, the plasmids used are shown in Table 1.

Table 1. Plasmids

In the following examples of the present invention, the applied strains are shown in Table 2.

Table 2. Strain

In the following examples of the present invention, the applied strains are shown in Table 3.

Table 3. Primers

In the table, underlines indicate restriction endonuclease cleavage points.

In the following examples of the present invention, the applied strains are shown in Table 4.

Table 4. Experimental reagents

2. Kits and tool enzymes

All restriction endonucleases and T4 ligases used for digestion and linearization were purchased from Bao Bioengineering (Dalian) Co., Ltd. rTaq and pfu enzymes were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

A small amount of plasmid extraction, gel recovery, product purification, and yeast genomic DNA extraction kits were purchased from Aisin Biotechnology (Hangzhou) Co., Ltd.

3. Culture medium

(1) E. coli growth medium (g/L)

LB: Yeast Extract 10, Peptone 20, NaCl 10.

The final concentration of Zeocin when formulating Zeocin-resistant medium was 25 μg/mL.

Add 2% agar powder when preparing solid medium.

(2) Yeast growth medium (g/L)

YPD: Yeast Extract 10, Peptone 20, Dextrose 20.

YPG: yeast extract 10, peptone 20, glycerol 20.

The final concentration of Zeocin when preparing Zeocin-resistant medium was 100 μg/mL.

Add 2% agar powder when preparing solid medium.

(3) Yeast shake flask fermentation medium

BSM: CaSO ₄ 0.46, PTM1 12mL/L, K ₂ SO ₄ 9.1, MgSO ₄ ·7H ₂ O 7.5, (NH ₄ ) ₂ SO _{4 7} , 0.2MK ₂ HPO ₄ /KH ₂ PO ₄ buffer (pH=5.5 ).

_{PTM1 : H3BO3 0.02 , Na2MoO4.2H2O0.2 , CuSO4.5H2O6} , _ZnSO4.7H2O20 , _{KI 0.08 , MnSO4.H2O3} , _CoCl2.6H2 O 0.5, FeSO ₄ ·7H ₂ O 65, biotin 0.2, H ₂ SO ₄ 5mL/L.

4. Cultivation method

(1) Seed cultivation

Pick a single colony from the plate stored at 4°C into 3 mL of YPG liquid medium, shake at 30°C and 220 rpm for 16 to 18 hours, take 1 mL of seed solution into 100 mL of YPG liquid medium, and culture at 30°C and 220 rpm for about 20 hours.

(2) Shake flask induction culture

The seed culture solution was transferred to a 50 mL sterile centrifuge tube, centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. The bacteria were resuspended in a 250 mL shake flask containing 25 mL of BSM medium (so that the OD ₆₀₀ was about 20), and induction culture was performed at 30° C. and 220 rpm. Take the time of adding methanol (1% (v/v)) as induction 0h, then add 1% (v/v) anhydrous methanol every 12h, and add 630μL 40g/L L-Met (24h) every 24h. Sterilized, the final concentration is 0.1%), the value of OD ₆₀₀ is measured every 24h. During the culture, the pH was adjusted with 5M sterile KOH to keep the pH at 5.5-6.0 for a total of 96 hours of induction.

5. Detection and analysis methods

(1) Determination of bacterial dry weight

Take a certain amount of bacterial liquid and dilute it by a certain number of times, and then use a visible spectrophotometer to measure its absorbance value at a wavelength of 600 nm, and use the sterile medium as a control for colorimetry. OD ₆₀₀ = OD reading×dilution factor, and the dry weight of bacteria was calculated from the standard curve.

Dry cell weight (DCW) (g/L)=0.2448×OD ₆₀₀ +1.2349 (R ² =0.994, OD ₆₀₀ ∈ [50,500].

(2) Determination method of intracellular SAM content

a. Sample processing

Take 1 mL of fermentation broth at 4°C, 12000 rpm, and centrifuge for 10 min. The supernatant was discarded, washed once with deionized water, resuspended in 1 mL of freshly prepared 10% trichloroacetic acid, and placed at 4°C for extraction for 2 h. Then, centrifuge for 10 min at 4°C and 12000 rpm, take the supernatant, filter with a 0.22 μm filter, and store at 4°C for detection. When the concentration is large, it should be diluted by an appropriate factor so that the peak area in HPLC is within the linear range.

b. Determination method

The chromatographic column is a strong cationic ion exchange column Thermo-BioBasic SCX (model 4.6 mm×250 mm, 5 μm). Mobile phase A was 5 mM ammonium formate (pH=4.0) and mobile phase B was 500 mM ammonium formate (pH=4.0). The flow rate was 1 mL/min, the injection volume was 10 μL, the column temperature was 30 °C, and the detection wavelength was 254 nm. The retention time of SAM is about 10.1 min.

(3) Intracellular SAM synthase enzyme activity assay method

a. Extraction of crude enzyme solution

Take 1 mL of fermentation broth, centrifuge for 10 min at 4°C, 12000 rpm. Discard the supernatant, wash once with deionized water, resuspend the resulting pellet in 1 mL lysis buffer, transfer to a 10 mL centrifuge tube, add an equal volume of acid-washed glass beads (0.5 mm in diameter), and place on a mediator shaker Shake for 30s, ice bath for 30s, repeat about 10 times, transfer the broken bacterial liquid into a 1.5mL centrifuge tube, centrifuge at 4°C, 12000rpm for 10min, and take the supernatant as the crude enzyme liquid.

b. Enzymatic reaction system

The enzymatic reaction was established according to the above system, and no L-Met was added as a blank control. The reaction system was carried out in a 37°C water bath. After the enzymatic reaction for 1 h, 500 μL of 20% perchloric acid solution was added, and the reaction was stopped at 4° C. for more than 30 min. 4°C, 12000rpm, centrifugation for 10min. Take the supernatant and filter it with a 0.22 μm filter for detection.

Calculation method of enzymatic activity: 1 enzymatic activity unit (U) is defined as the enzymatic activity of SAM synthase corresponding to the conversion of 1 μmoL of SAM within 1 h at 37°C.

Example 1. Construction of enhanced expression vector for sol3 and zwf1 genes

pAOX-SSN construction method:

According to the genomic sequence of GS115, a sequence for homologous recombination was found, and about 700 bp of each of the two open reading frames (ORF) was taken as the homology arm. The homology arms were named RH and DDKC, respectively. The CBS5' fragment on the plasmid pCmG (Qiao Xuefeng, optimized the synthesis of S-adenosylmethionine by Pichia pastoris [D]. Shanghai: East China University of Science and Technology, 2013) was excised with XhoI and SpeI, and the upstream same The source arm RH is connected to the original position of CBS5'. The PADH2-vgb-TT unit (Qiao Xuefeng; using marker-free gene manipulation to optimize the synthesis of S-adenosylmethionine by Pichia pastoris [D]; Shanghai: East China University of Science and Technology, 2013) and DDKC were used for overlapping PCR to obtain the entire DNA. Fragment. The PGAP-CBS ORF fragment on the plasmid with the RH homology arm was excised with PstI and EcoRI, and the obtained overlapping fragment with the vgb gene was ligated between the PstI and EcoRI sites to obtain the plasmid pCmG (R-A-D). The construction process is shown in Figure 5.

Taking the plasmid pCmG(R-A-D) as the starting plasmid, the AOX promoter and the gln1 gene were connected into a fragment by overlapping PCR, digested with SpeI and NotI, and connected to pCmG(R-A-D), and the obtained plasmid was named pAOX-gln, as shown in Figure 6 .

Using pAOX-gln as the starting plasmid, the PAOX with the restriction site was obtained by PCR amplification. At the same time, the fragment and the plasmid were cut with SpeI and NotI, and then connected to obtain pAOX(SSN). The construction process is shown in Figure 7.

The Zwf1 gene was amplified by using Zwf1F and Zwf1R as primers and G12' genomic DNA as a template.

Sol3 gene was obtained by amplification with Sol3F and Sol3R and G12' genomic DNA as template.

Using plasmid pAOX-SSN as the starting vector, the target gene and plasmid pAOX-SSN were double digested with restriction endonucleases SacII and NotI. After purification and recovery, the recombinant plasmids obtained by ligating with T4 ligase were named pAOX-zwf1 and pAOX respectively. -sol3, the construction process is shown in Figure 1. The obtained plasmid was constructed, and the sequence was verified by enzyme digestion and sequencing.

Embodiment 2, the construction of recombinant bacteria

The plasmid pCmG (Qiao Xuefeng. Using marker-free gene manipulation to optimize the synthesis of S-adenosylmethionine [D] by Pichia pastoris; Shanghai: East China University of Science and Technology, 2013.) The weakened promoter replacement unit of ZeoR was electroporated to transform SAM high-producing strain DS16. Recombinant bacteria G/CmG were obtained by screening on a plate containing Zeocin. Then, through the homologous recombination between the forward repeat sequences at both ends of the marker recovery unit (MazF-ZeoR), the recovery of the selection marker is realized, so that the recombinant strain G/CmG no longer has ZeoR, and a new recombinant strain G/CG, namely G12 ' (Figure 8). The whole process can also be found in Qiao Xuefeng; Using marker-free gene manipulation to optimize the synthesis of S-adenosylmethionine by Pichia pastoris [D]; Shanghai: East China University of Science and Technology, 2013.

The constructed recombinant plasmid was linearized by XhoI/EcoRI double enzyme digestion, purified and recovered the target fragment, and the target fragment was transferred into Pichia pastoris G12', through the RH-DDKC site, with ADRH and DDKC in the G12' genome Integration into the G12' genome occurred by homologous recombination (Figure 2), and single clones were picked on Zeocin-resistant plates.

According to the above steps, recombinant bacteria G12'/AOX-zwf1 and G12'/AOX-sol3 were obtained.

Embodiment 3, the enzyme activity and output of recombinant bacteria

The recombinant strains G12'/AOX-zwf1 and G12'/AOX-sol3 were fermented in shake flasks, and the starting strain G12' was used as a control strain to investigate the effects of enhanced expression of zwf1 and sol3 genes on bacterial growth and SAM synthesis.

The initial concentration of induced bacteria was controlled at the same level, and methanol was added as the zero point of induction. Methanol was added every 12h until the final concentration of methanol in the fermentation broth was 1%, and 1% L-Met (w/ 24h) was added. v), the whole process was adjusted to pH 5.5-6.0 with 5M KOH for a total of 96 hours of induction. Samples were taken every 24 hours to measure OD ₆₀₀ , and the growth curve was obtained, as shown in Figure 3 . The growth trend of the recombinant bacteria was consistent with that of the control bacteria G12', indicating that the overexpression of each gene did not affect the normal growth of the bacteria.

The SAM production and SAM synthase activity of the recombinant bacteria were determined after 96 hours of induction and culture (Fig. 4). The SAM yield of G12'/AOX-sol3 was 182.17 mg/g DCW, which was 14.5% higher than that of the original strain G12' (159.11 mg/g DCW). The SAM yield of G12'/AOX-zwf1 was 178.87 mg/g DCW, which was comparable It is 12.4% higher than the starting bacteria.

Investigating the corresponding SAM synthase activity, the highest enzyme activity of G12'/AOX-zwf1 was 442.72U/g DCW, which was 26.0% higher than that of the starting strain G12' (351.05U/g DCW). G12'/AOX-sol3 The enzyme activity also reached 417.62U/g DCW, an increase of 18.7% compared with the starting bacteria.

The above results indicate that overexpression of sol3 and zwf1 genes can significantly increase the enzymatic activity of SAM synthase and promote SAM synthesis.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (7)

1. a method for producing S-adenosylmethionine, wherein the method comprises: (1) An exogenous 6-phosphate glucose dehydrogenase expression cassette was introduced into the genome of the S-adenosylmethionine-producing strain; the S-adenosylmethionine-producing strain was (a) inserted into SAM synthase gene ds16, and a strain with weakened β-cystathionine synthase gene; or (b) on the basis of strain (a), a strain with the selection marker excluded; (2) culturing the strain prepared in step (1) to produce S-adenosylmethionine; the culturing method includes: inducing culture under the conditions of 30±2° C. and 220±50 rpm; adding methanol for the first time to a final concentration of 1± After 0.2%, methanol was added every 12±2h until its final concentration was 1±0.2% by volume; L-Met was added every 24±4h, and a total of 96±12h was induced. 2. The method of claim 1, wherein the 6-phosphate glucose dehydrogenase expression cassette comprises the following operably linked elements: AOX1 promoter, a gene encoding 6-phosphate glucose dehydrogenase, Transcription termination sequence. 3. The method of claim 1, wherein in step (2), the method for culturing comprises: inducing culture at 30° C. and 220 rpm; after adding methanol to a final concentration of 1% for the first time, replenishing every 12h Methanol was added to the final concentration of 1% by volume; L-Met was added every 24h for a total of 96h induction. 4. an improved S-adenosylmethionine production strain, characterized in that, an exogenous 6-phosphate glucose dehydrogenase expression cassette has been introduced into the genome of the strain; the S-adenosine The methionine-producing strain is (a) a strain in which the SAM synthase gene ds16 is inserted and the β-cystathionine synthase gene is weakened; or (b) a strain in which the selection marker is excluded on the basis of (a) strains . 5. bacterial strain as claimed in claim 4, is characterized in that, described 6-phosphate glucose dehydrogenase expression cassette comprises following operably linked element: AOX1 promoter, the gene encoding 6-phosphate glucose dehydrogenase, Transcription termination sequence. 6. the purposes of 6-phosphate glucose dehydrogenase, is characterized in that, is used for promoting S-adenosylmethionine production strain to produce S-adenosylmethionine; Described S-adenosylmethionine production strain It is (a) a strain in which the SAM synthase gene ds16 is inserted and the β-cystathionine synthase gene is attenuated; or (b) a strain in which the selection marker is excluded based on the (a) strain; the production includes: Induction culture was carried out at 30±2°C and 220±50rpm; after the first addition of methanol to a final concentration of 1±0.2%, methanol was added every 12±2h to a final concentration of 1±0.2% by volume; every 24±0.2% L-Met was supplemented for 4h, and the cells were induced for 96±12h. 7. A kit for producing S-adenosylmethionine, characterized in that the kit comprises the improved S-adenosylmethionine producing strain according to claim 4 or 5.

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