CN105602966A - Gene of coded 6-phosphogluconate dehydrogenase and application thereof - Google Patents

Abstract

The invention provides a gene of coded 6-phosphogluconate dehydrogenase and application thereof. The nucleotide sequence is shown in SEQ ID NO:1. The 6-phosphogluconate dehydrogenase gene pgdhD2 is derived from Methylobacterium sp. MB200 and has the L-serine inhibiting effect. The Methylobacterium sp. MB200 is used as an original strain, pgdh gene deletion mutation DMB is built through a homologous double-crossover method, an L-serine high-yield mutant strain with the L-serine producing capacity greatly higher than that of original strain Methylobacterium sp. MB200 is obtained, and the tolerated D-serine concentration of the L-serine high-yield mutant strain is greatly higher than that of the original strain. The pgdh gene deletion mutation stain has the good application prospect in production of L-serine through a fermentation method.

Description

A gene encoding 6-phosphogluconate dehydrogenase and its application

technical field

The invention belongs to the technical field of genetic engineering, in particular to a gene encoding 6-phosphogluconate dehydrogenase and its application.

Background technique

L-serine has important physiological functions and application value, and is widely used in the fields of medicine, food and chemical industry. At present, the production methods of L-serine mainly include chemical synthesis, proteolysis, enzymatic transformation and microbial fermentation. The production of L-serine by microbial fermentation is a green and environmentally friendly production method, but due to the feedback inhibition regulation, it is difficult for microorganisms to accumulate L-serine in large quantities, so the yield of L-serine produced by microbial fermentation is relatively low. application of this method.

The method for removing the feedback inhibition regulation of L-serine and improving the yield of L-serine produced by fermentation method, one is to obtain the key enzyme that has anti-L-serine feedback inhibition regulation through evolutionary transformation of key enzyme genes in the L-serine biosynthesis pathway. Enzyme, release the feedback inhibition of L-serine, construct L-serine high-yielding strain; the second is to transform the key enzymes of non-synthetic pathways such as EMP pathway, HMP pathway, L-serine conversion pathway, transport pathway, etc., which can also reduce or relieve L-serine Serine Feedback Inhibition: Construct related gene mutant L-serine high-yielding strains to relieve the feedback inhibition of L-serine.

Releasing the feedback inhibition of L-serine on the key enzymes of the synthetic pathway is the key to constructing L-serine high-yielding strains, and genetic evolution is the main means to modify the key enzymes of the metabolic pathway and their gene expression regulatory sequences. Relevant studies have been carried out using molecular biology techniques to reduce the sensitivity of key enzymes in the synthetic pathway to L-serine, so that engineered strains have anti-L-serine feedback inhibition regulation to improve the accumulation of L-serine and improve the biosynthesis of L-serine yield. For example, the Chinese patent application (publication number 103436504A) discloses that the strain Corynebacterium glutamicum SYPS-062, which produces L-serine, is used as the starting strain, and the 3-phosphoglyceric acid that catalyzes the first step reaction of its biosynthetic pathway is desorbed by means of genome engineering. Evolutionary modification of hydrogenase to achieve constitutive and stable expression of 3-phosphoglycerate dehydrogenase mutants resistant to L-serine feedback inhibition, and obtaining stable expression of 3-phosphoglycerate dehydrogenase mutants resistant to L-serine feedback inhibition , and constructed a high-yielding strain of L-serine. Chinese patent (publication number 1609208) discloses the substitution of the 349th glycine or the 372nd threonine of Escherichia coli 3-phosphoglycerate dehydrogenase (3-PGDH), and the obtained mutant is sensitive to L-serine lower for better results. Chinese patent application (publication number 102433312A) also uses site-directed mutation to mutate the 344th histidine to alanine and the 346th asparagine to alanine in 3-phosphoglycerate dehydrogenase (3-PGDH) , the enzyme activity of the mutant is not inhibited by L-serine and the enzyme activity can be retained to a large extent.

Methylobacterium sp.MB200 is an L-serine-producing strain isolated from the residue in the biogas digester. It is different from other serine-producing strains because it can use non-C-C bond low-carbon complexes such as methanol to grow. Metabolic pathways of microorganisms such as Escherichia coli and Corynebacterium glutamicum. Studies have found that in methylotrophic bacteria such as Methylobacterium extorquensAM1, there is a complex metabolic network as shown in Figure 1. It can be seen from Figure 1 that in methylotrophic bacteria, methanol is first oxidized to methylenetetrahydrofolate, and then reacts with glycine to generate L-serine. Methyl assimilation is mainly realized through the C1 pathway and the serine cycle, and the C1 pathway and the serine cycle are connected with the EMP pathway, the HMP pathway, and the TCA cycle through different carbon compounds, coenzymes, ATP, etc., forming a mutual influence and interaction. An implicated and regulated metabolic network.

The L-serine biosynthesis of the methylobacterium Methylobacterium sp.MB200 is in such a metabolic network, and it is also strictly regulated. The product L-serine can feedback inhibit the activity of certain enzymes in the metabolic network, so that the L-serine biosynthesis is in the Inhibited state. In order to relieve the feedback regulation of the product on the key enzymes in the synthetic pathway, the usual strategy is to breed anti-metabolic regulation mutants, and the selection of product structural analogue-resistant mutants is the most common method of this breeding strategy. Use the plasmid transposon insertion technology to construct a mutant library, screen out D-serine (L-serine structural analogue) resistant mutants, and clone the sequence of the inserted fragment, and analyze the mutated gene, the enzyme encoded by the gene That is, there may be a feedback regulation effect of L-serine. By analyzing the relationship between the enzyme activity and its function and L-serine, it can be verified whether the enzyme has the feedback inhibition effect. If the enzyme encoded by the obtained gene has the L-serine feedback inhibition effect, then the gene is evolved and modified to construct a mutant strain, and a high-production strain of L-serine can be established. In addition, methylotrophic bacteria have unique metabolic pathways and complex metabolic networks. EMP, HMP, TCA, and C1 pathways are all inextricably linked with the serine cycle. Through the above methods, it is possible to clone L-serine Genes for enzymes with feedback inhibition effects (such as 3-PGDH). Through constructing recombinant bacteria and enzyme activity experiments, analyzing related gene functions and their relationship with L-serine production, constructing related gene mutant strains, removing methylotrophic bacteria L-serine feedback inhibition regulation, and obtaining L-serine high-yielding strains. It provides new ideas and new ways for constructing L-serine high-yielding strains.

Contents of the invention

Aiming at the problems of feedback inhibition regulation and relatively low yield of L-serine produced by the fermentation of methylobacterium sp.MB200, the present invention uses plasmid transposon insertion technology to construct a mutant library of methylobacterium sp.MB200, and screens out A mutant with D-serine resistance, cloned the 6-phosphogluconate dehydrogenase (6-PGDH) gene with L-serine inhibitory effect, and constructed a pgdh gene-deficient L-serine high-yielding strain, relieved The inhibitory effect of 6-PGDH on the production of L-serine during the metabolic process of methylotrophic bacteria, thereby increasing the production of L-serine.

The technical scheme provided by the invention is:

A gene pgdhD2 encoding 6-phosphogluconate dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO:1.

In the technical scheme of the present invention, the gene pgdhD2 encoding 6-phosphogluconate dehydrogenase, the DNA in its nucleotide sequence SEQ ID NO: 1 starts the initiation codon ATG from the first nucleotide at the 5' end There is a complete open reading frame (ORF) until the 993rd nucleotide ends with the stop codon TGA, and the 1-3 nucleotides from the 5' end are the start codon ATG of the pgdhD2 gene, starting from the 5' The 991-993 nucleotides at the end are the stop codon TGA of the pgdhD2 gene.

In the technical scheme of the present invention, the gene pgdhD2 of described encoding 6-phosphogluconate dehydrogenase has an open reading frame (ORF) in its nucleotide sequence SEQIDNO: 1, has a total of 993 nucleotides, and can encode A protein consisting of 330 amino acids.

The present invention also provides a protein encoded by the gene pgdhD2 encoding 6-phosphogluconate dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO:2.

An expression vector comprising the gene pgdhD2 sequence encoding 6-phosphogluconate dehydrogenase.

A mutant cell line of Methylobacteriumsp.MB200 lacking the sequence encoding 6-phosphogluconate dehydrogenase gene pgdhD2.

The present invention also provides the application of the gene pgdhD2 encoding 6-phosphogluconate dehydrogenase in constructing the L-serine-producing mutant strain lacking the gene pgdhD2 encoding 6-phosphogluconate dehydrogenase.

The concrete operation of the method that the present invention adopts is as follows:

(1) Cloning and analysis of the D-serine-tolerant mutant gene of the methylotrophic bacteria Methylobacteriumsp.MB200: the mutant library of the methylobacteriumsp.MB200 was constructed by plasmid transposon insertion technology, and the D- Serine-resistant mutants. Extract the total DNA from the mutant, connect it to the cloning vector pMD-18T after enzyme digestion, and introduce it into Escherichia coli competent cell DH5α, use double resistance to pick transformants on the plate, and extract the plasmid, after enzyme digestion verification Further sequencing, Blast sequence alignment and analysis in NCBI showed that the insert of the mutant was the D-serine-tolerant 6-phosphogluconate dehydrogenase gene of the methylotrophic bacteria Methylobacteriumsp.MB200.

(2) Cloning, expression and enzyme activity analysis of 6-phosphogluconate dehydrogenase gene: primers were designed according to the 6-phosphogluconate dehydrogenase gene sequence of Methylobacteriumsp.MB200, and the total DNA of M.sp.MB200 was used as a template Perform PCR to obtain the pgdh gene fragment (with EcoRI and PstI restriction sites). The obtained pgdh fragment and the vector pETBlue-2 were digested with EcoRI and PstI respectively, purified, ligated, and transformed into competent cells of Escherichia coli BL21(DE3)pLysS to obtain recombinant BL21(DE3)pLysS/pETBlue- 2-pgdh. After the recombinant protein PGDH is expressed under suitable conditions, it is purified with a nickel column.

(3) Determination of recombinant protein PGDH enzyme activity and functional verification: Determination of recombinant protein PGDH enzyme activity. In a reaction system containing 100mM Tris-HCl (pH8.0), 10mM MgCl ₂ , 3mM NADP ⁺ , and 3mM 6-PG, add appropriately diluted enzyme solution, and measure and calculate the change of NADPH concentration under the reaction condition of 40°C. The enzyme activity is defined as 1min catalytic The amount of enzyme that produces 1mM NADPH is one enzyme activity unit U.

Add different concentrations of L-serine to the enzyme activity assay system, measure the PGDH enzyme activity at different L-serine concentrations, analyze the effect of L-serine on the enzyme activity of recombinant protein PGDH, and verify the 6-phosphogluconate dehydrogenase gene Inhibitory effect of L-serine.

(4) Construction of methylotrophic bacteria Methylobacteriumsp.MB2006-phosphogluconate dehydrogenase gene deletion mutant: using methylotrophic bacteria Methylobacteriumsp.MB200 as the starting strain, the pgdh gene deletion mutant DMB was constructed by homologous double exchange method, A pgdh gene deletion mutant strain with high L-serine production ability was obtained. The function of the 6-phosphogluconate dehydrogenase gene was verified by analyzing the production of L-serine and resistance to D-serine of the pgdh gene deletion mutant strain DMB and the enzyme activity.

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention utilizes plasmid transposon insertion technology to construct a mutant library of methylotrophic bacteria Methylobacteriumsp.MB200, from which the mutants with D-serine resistance are screened out, and the method for encoding 6-phosphate is obtained by sequencing analysis and cloning. The pgdh gene of gluconate dehydrogenase (PGDH), and through the construction of recombinant bacteria and enzyme activity experiments, it is shown that L-serine has an inhibitory effect on PGDH enzyme activity. In the present invention, the methylotrophic bacteria Methylobacteriumsp.MB200 is used as the starting strain, and the pgdh gene deletion mutant strain DMB is constructed by a homologous double exchange method, and a high-yield L-serine mutant strain is obtained whose ability of producing L-serine is much stronger than that of the starting strain Methylobacteriumsp.MB200 DMB can tolerate 5 mg/mL of D-serine, which is much higher than the ability of the starting strain to tolerate the concentration of D-serine not exceeding 1 mg/mL.

2. The present invention obtains a high-yielding strain of L-serine by constructing a pgdh gene deletion mutant strain DMB, releasing the L-serine feedback inhibition regulation of methylotrophic bacteria Methylobacteriumsp. way.

Description of drawings

Accompanying drawing 1 is the metabolic network of methylotrophic bacteria such as MethylobacteriumextorquensAM1;

Accompanying drawing 2 is the growth situation of each bacterial strain of methylotrophic bacteria Methylobacteriumsp.MB200, pgdh gene deletion mutant, complementing bacteria AMB and overexpressing bacteria EMB on the D-serine gradient plate;

Accompanying drawing 3 is the DNS thin-layer chromatogram of L-serine produced by the fermentation liquid of each strain of methylotrophic bacteria Methylobacteriumsp.MB200, pgdh gene deletion mutant, complementing bacteria AMB and overexpressing bacteria EMB;

Explanation of reference signs:

A-M.sp.MB200; B-deletion mutant DMB; C-replenishment strain AMB; D-overexpression strain EMB; 1-standard glycine; 2-standard serine; 3-M.sp.MB200; 4-deletion mutant DMB; 5-replenishment strain AMB; 6-overexpression strain EMB.

detailed description

The specific embodiments of the present invention will be described in detail below in conjunction with the examples, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations thereof such as "includes" or "includes" and the like will be understood to include the stated elements or constituents, and not Other elements or other components are not excluded.

Embodiment 1 :

1.1 Cloning and analysis of the D-serine tolerance mutant gene of methylotrophic bacteria Methylobacteriumsp.MB200

1.1.1 The mutant library of methylobacterium sp.MB200 was constructed by plasmid transposon insertion technology, and the mutant D-2 with D-serine resistance was screened out.

1.1.2 Extract the total DNA from the mutant D-2, connect it to the cloning vector pMD-18T after enzyme digestion, and introduce it into the competent E. Plasmids were further sequenced after digestion and verification, and Blast sequence alignment and analysis were performed in NCBI. As a result, the obtained DNA fragment was 1875bp, and the sequence had the highest similarity with the 6-phosphogluconate dehydrogenase gene of MethylobacteriumpopuliBJ001, with a consistency of 89%. See Table 1 for details.

Mutant DNA fragments cloned in table 1

1.2 Bioinformatics analysis of inserted DNA fragments in mutant strain D-2

The DNA fragment obtained from the mutant strain D-2 is 1875bp, and the ORF of the mutant D-2 DNA fragment is searched using ORFFinder on NCBI, which contains an ORF of 993bp. The ORF sequence is 97% consistent with the 6-phosphogluconatedehydrogenase of MethylobacteriumpopuliBJ001, MethylobacteriumextorquensAM1, MethylobacteriumchloromethanicumCM4 at the amino acid level, and the similarity is 98%. It can be seen that this sequence encodes the 6-phosphogluconated dehydrogenase of M.sp.MB200 Hydrogenase.

It is predicted that the protein contains 330 amino acid residues, and the amino acids with relatively large content and their contents are: Gly12.4%, Ala10.9%, Arg9.4%, Leu8.8%, Asp7.6%, Glue7.6%. The molecular composition is C1572H2479N463O481S11. The molecular weight is 35913.4, and the theoretical pI=5.22.

It has no obvious hydrophilicity and hydrophobicity, no transmembrane structure, and contains a pfamNADbinding domain.

1. Cloning and expression of 36-phosphogluconate dehydrogenase gene

1.3.1 Cloning of 6-phosphogluconate dehydrogenase gene

Primers were designed according to the sequence of the Methylobacteriumsp.MB2006-phosphogluconate dehydrogenase gene, and PCR was performed using the total DNA of M.sp.MB200 as a template to obtain the pgdh gene fragment (with EcoRI and PstⅠ restriction sites). The obtained pgdh fragment and the vector pETBlue-2 were digested with EcoRI and PstI respectively, purified, ligated, and transformed into competent cells of Escherichia coli BL21(DE3)pLysS to obtain recombinant BL21(DE3)pLysS/pETBlue- 2-pgdh.

1.3.2 Expression and purification of recombinant protein PGDH

After the recombinant protein is expressed under suitable conditions, it is purified with a nickel column.

1.4 Enzymatic activity of recombinant protein PGDH

1.4.1 Determination of enzyme activity

The reaction system is 300uL, which contains 100mM Tris-HCl (pH8.0), 10mM MgCl ₂ , 3mM NADP ⁺ , 3mM 6-PG, 50uL of appropriately diluted enzyme solution is added, supplemented with water, and mixed well. Measure the OD value at 40°C and 340nm, and record once every 10S for a total of 10min. The change of NADPH concentration can be calculated from the change value of OD.

Enzyme activity definition: Under optimal conditions, the amount of enzyme that catalyzes the production of 1mM NADPH in 1 minute is one enzyme activity unit U.

1.4.2 Effect of L-Ser concentration on activity of recombinant protease

Add different concentrations of L-serine to the enzyme activity assay system, and measure the PGDH enzyme activity under different L-serine concentrations. The results show that the addition of L-serine can inhibit the enzyme activity, and the inhibitory effect is strengthened with the increase of the concentration of L-serine. When the concentration reaches 500mM, the relative enzyme activity decreases to about 30%.

1.5 Verify the function of 6-phosphogluconate dehydrogenase gene and its L-serine inhibitory effect in Methylobacteriumsp.MB200

1.5.1 Construction of pgdh gene deletion mutants, complementation bacteria and overexpression bacteria

The pgdh gene deletion mutant strain DMB was constructed by the method of homologous double crossover, based on which the anaplerotic strain AMB and the pgdh gene overexpression strain EMB were constructed by the method of three-parent conjugation through the vector pCM80.

1.5.2 Verification of the function of 6-phosphogluconate dehydrogenase gene in pgdh gene deletion mutant bacteria, supplementary bacteria and overexpression bacteria

Taking the pgdh gene deletion mutant strain DMB, replenishment strain AMB, overexpression strain EMB and the original strain M.sp. Validation of the function of the 6-phosphogluconate dehydrogenase gene.

1.5.2.1 The tolerance of each strain to D-serine

The tolerance of each strain to D-serine was studied experimentally, and the results are shown in FIG. 2 . It can be seen from the figure that the starting strain and the overexpressing bacteria can tolerate D-serine not exceeding 1 mg/mL, the deletion mutant can tolerate D-serine at 5 mg/mL, which is more than 5 times that of the starting strain, and the complemented strain can tolerate D-serine - Serine is 1.2mg/mL, slightly higher than the starting strain.

1.5.2.2 The ability of each strain to produce L-serine

After each strain was fermented and cultured in a resting cell system, DNS chromatography was used to semi-qualitatively analyze the ability to produce L-serine. The DNS chromatogram is shown in FIG. 3 . The results in the figure show that the ability of the strains to produce L-serine is the strongest in the deletion mutant DMB, the complementary strain AMB and the overexpression strain EMB are basically the same, and the M.sp.MB200 is the weakest.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling others skilled in the art to make and use various exemplary embodiments of the invention, as well as various Choose and change. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims (7)

1. the encode gene pgdhD2 of 6-phosphogluconate dehydrogenase, is characterized in that: its nucleosidesAcid sequence is as shown in SEQIDNO:1.

2. the gene pgdhD2 of coding 6-phosphogluconate dehydrogenase according to claim 1, its spyLevy and be: the DNA in its nucleotide sequence SEQIDNO:1 is initial close since the 1st nucleotides of 5 ' end993 nucleotides of numeral ATG to the finish with terminator codon TGA, have a complete open readingFrame (ORF) is the initiation codon ATG of pgdhD2 gene from the 1-3 position nucleotides of 5 ' end, from 5 'The 991-993 position nucleotides of end is the terminator codon TGA of pgdhD2 gene.

3. the gene pgdhD2 of coding 6-phosphogluconate dehydrogenase according to claim 1, itsBe characterised in that: the ORFs (ORF) in its nucleotide sequence SEQIDNO:1, has 993Nucleotides, the protein that codified is made up of 330 amino acid.

4. the gene by the arbitrary described coding 6-phosphogluconate dehydrogenase of claim 1-3The protein of pgdhD2 coding, is characterized in that: its amino acid sequence is as shown in SEQIDNO:2.

5. an expression vector that comprises coding 6-phosphogluconate dehydrogenase gene pgdhD2 sequence.

6. one kind lacks the clone of coding 6-phosphogluconate dehydrogenase gene pgdhD2 sequence.

7. according to the gene of the arbitrary described coding 6-phosphogluconate dehydrogenase of claim 1-3PgdhD2 is prominent at the product Serine that builds disappearance coding 6-phosphogluconate dehydrogenase gene pgdhD2Application in modification bacterial classification.