CN116445381A - Ammonium-resistant nitrogen fixation microorganism over-expressing ald gene, construction method and application thereof - Google Patents

Abstract

The invention discloses an ammonium-resistant nitrogen fixation microorganism over-expressing an ald gene, a construction method and application thereof. The ammonium-resistant nitrogen-fixing microorganism is obtained by overexpressing an ald gene from a paenibacillus microorganism in the paenibacillus microorganism. The constructed ammonium-resistant nitrogen fixation microorganism can carry out biological nitrogen fixation under the conditions of limited ammonium and high ammonium, breaks through the inhibition effect of the high ammonium condition on the biological nitrogen fixation, and has wide application prospect in agricultural production.

Description

Ammonium-resistant nitrogen fixation microorganism over-expressing ald gene, construction method and application thereof

Technical Field

The invention relates to the fields of genetic engineering and microorganisms, in particular to an ammonium-resistant nitrogen fixation microorganism over-expressing an ald gene, and a construction method and application thereof.

Background

Nitrogen is the largest element necessary in plant production. The chemical nitrogenous fertilizer plays an important role in guaranteeing grain production, vegetable planting and fruit tree cultivation in China. However, excessive chemical nitrogenous fertilizer is applied for a long time, so that soil acidification and salinization are caused, and the microbial activity of the soil is reduced, so that the soil becomes an important limiting factor for sustainable development of agriculture.

The nitrogen-fixing microorganism can reduce nitrogen in the air into ammonium by utilizing in-vivo nitrogen-fixing enzyme at normal temperature and normal pressure, so as to be used for plant growth. However, nitrogen fixation efficiency is affected by the concentration of ammonium, i.e., nitrogen fixation at limited or low ammonium concentrations, which is typically greater than 5mM or more inhibits nitrogen fixation. In barren soil, nitrogen fixing microorganisms have high nitrogen fixing efficiency, while in fertile soil, only grow but do not fix nitrogen. Therefore, the microorganism for fixing nitrogen under the condition of high ammonium is obtained, and has important application value in agricultural production.

Disclosure of Invention

The invention aims to provide an ammonium-resistant nitrogen fixation microorganism over-expressing an ald gene, and a construction method and application thereof.

The invention is characterized in that: the ald gene is present in some microorganisms, and the alanine dehydrogenase encoded by the ald gene catalyzes the synthesis and decomposition of alanine, and the precursor for the synthesis of alanine is pyruvic acid, thus acting as an intermediate bridge in carbon-nitrogen metabolism. The invention discovers for the first time that in the nitrogen fixation bacillus, the high ammonium nitrogen fixation phenomenon is related to alanine dehydrogenase.

To achieve the object of the present invention, in a first aspect, the present invention provides an ammonium-resistant nitrogen-fixing microorganism overexpressing an ald gene, which is obtained by overexpressing an ald gene from a Paenibacillus microorganism in a Paenibacillus (Paenibacillus) microorganism.

The pathway of overexpression may be selected from the following 1) to 6), or an optional combination:

1) Enhanced by introducing a plasmid having the gene;

2) Enhanced by increasing the copy number of the gene on the chromosome;

3) Enhanced by altering the promoter sequence of said gene on the chromosome;

4) Enhanced by operably linking a strong promoter to the gene;

5) Enhanced by the introduction of enhancers;

6) Enhanced by the use of a gene or allele having a high activity encoding an alanine dehydrogenase.

The ald gene may be derived from Paenibacillus polymyxa (Paenibacillus polymyxa), paenibacillus azotidis (Paenibacillus sabinae), and the like.

In one embodiment of the invention, the ald gene is derived from Paenibacillus polymyxa WLY78, which has the nucleotide sequence:

i) A nucleotide sequence shown in SEQ ID NO. 1;

ii) the nucleotide sequence shown in SEQ ID NO. 1 is substituted, deleted and/or added with one or more nucleotides and expresses the same functional protein;

iii) A nucleotide sequence which hybridizes to the sequence shown in SEQ ID No. 1 and expresses the same functional protein under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or in a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution; or (b)

iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

The amino acid sequence of the alanine dehydrogenase encoded by the ald gene is shown in SEQ ID NO. 4.

Alternatively, the ald gene overexpression is achieved by: TGTCAT in the transcription regulatory factor GlnR binding site (sequence of GlnR binding site: TGTCATTTTACATGACA) of the ald gene promoter region (SEQ ID NO: 5) was replaced with GGATCC (BamH I cleavage site).

Further, the starting strain may be Paenibacillus polymyxa, preferably Paenibacillus polymyxa WLY78.

In a second aspect, the invention provides a method for constructing an ammonium-resistant nitrogen-fixing microorganism over-expressing an ald gene, comprising the steps of:

(1) PCR amplification is carried out by using primers aldPF and aldR (SEQ ID NO:2 and 3) and using Paenibacillus polymyxa (Paenibacillus polymyxa) WLY78 genome as a template to obtain a fragment with a length of 1329 bp; cutting the glue to recover fragments by using a Tiangen glue recovery kit;

(2) The plasmid pHY300PLK was digested with BamHI and HindIII to obtain an enzyme fragment;

(3) Assembling the recovered fragment in the step (1) and the enzyme-digested fragment in the step (2) through Gibson to obtain a recombinant vector pHY78ald;

(4) Transforming the recombinant vector pHY78ald into competent cells of the Paenibacillus polymyxa WLY78, and screening strains with tetracycline resistance.

Preferably, the conversion of step (4) is a shock conversion.

In a third aspect, the invention provides the use of the ald gene for nitrogen fixation under high ammonium conditions in the induction of paenibacillus microorganisms.

The application comprises: and (3) using a genetic engineering means to enable the microorganism to overexpress the ald gene so as to induce the paenibacillus microorganism to fix nitrogen under the high ammonium condition.

The ald gene is derived from a microorganism of the genus Paenibacillus, preferably Paenibacillus polymyxa, paenibacillus azoxygenus, more preferably Paenibacillus polymyxa WLY78.

The starting strain may be Paenibacillus polymyxa, preferably Paenibacillus polymyxa WLY78.

In the application, the high ammonium condition refers to NH in the environment of the microorganism ₄ ⁺ The concentration of (C) is 30-400 mM.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the invention provides an ammonium-resistant nitrogen fixation microorganism which can carry out biological nitrogen fixation under the conditions of limited ammonium and high ammonium, successfully endows the strain with high ammonium nitrogen fixation capacity by overexpressing the ald gene in Paenibacillus polymyxa WLY78, breaks through the inhibition effect of the high ammonium condition on the biological nitrogen fixation, and has wide application prospect in agricultural production.

Drawings

FIG. 1 shows a comparison of the activity of the enzyme at different ammonium concentrations of wild-type strain (WT) and WT/ald strain according to the preferred embodiment of the invention. Wherein, 1: wild-type strain (WT), 2: WT/ald strain.

FIG. 2 is a comparison of the activity of the enzyme nitrogen fixation enzyme under conditions of no ammonium and high ammonium for wild type strain (WT) and WT/27ald strain in the preferred embodiment of the invention.

FIG. 3 shows comparison of the activity of the enzyme nitrogen fixation enzyme under ammonium free and high ammonium conditions for wild type strain (WT) and MPald strain in the preferred embodiment of the invention.

In fig. 2 and 3, -N: ammonium-free medium, +n: high ammonium medium.

Detailed Description

The invention aims to provide an ammonium-resistant nitrogen fixation microorganism over-expressing an ald gene, and a construction method and application thereof.

The invention adopts the following technical scheme:

the invention provides an ammonium-resistant nitrogen-fixing microorganism over-expressing an ald gene, which is obtained by recombining the ald gene from paenibacillus polymyxa and an expression vector and then converting the ald gene into the nitrogen-fixing microorganism, wherein the nucleotide sequence of the ald gene is shown as SEQ ID NO. 1.

Further, the microorganism is a nitrogen-fixing paenibacillus microorganism.

Further, the invention provides a construction method of the ammonium-resistant nitrogen-fixing microorganism by taking Paenibacillus polymyxa as an example, which comprises the following steps:

(1) PCR amplification using primers aldPF and aldR (SEQ ID NO:2 and 3) and Paenibacillus polymyxa WLY78 genome as template to obtain 1329bp long expression cassette; cutting the glue to recover fragments by using a Tiangen glue recovery kit;

(2) The plasmid pHY300PLK was digested with BamHI and HindIII to obtain an enzyme fragment;

(3) Assembling the recovered and purified fragment with the digested and purified pHY300PLK by Gibson assembly master mix (New England Biolabs) to obtain a recombinant vector pHY78ald;

(4) The recombinant vector pHY78ald is transformed into Paenibacillus polymyxa WLY competent cells by electric shock, strains with tetracycline resistance are obtained by screening, and the ald gene overexpression strain WT/ald is obtained by PCR verification and sequencing confirmation.

In a second aspect, the present invention provides the use of the ald gene of SEQ ID NO. 1 for inducing nitrogen fixation in a paenibacillus microorganism under high ammonium conditions by allowing an expression vector comprising the ald gene in the microorganism to induce nitrogen fixation in the paenibacillus microorganism under high ammonium conditions.

The Paenibacillus microorganism is preferably Paenibacillus polymyxa.

The high ammonium condition refers to NH in the environment where the microorganism is located ₄ ⁺ The concentration of (C) is 30-400 mM.

The ammonium-resistant nitrogen-fixing microorganism over-expressing the ald gene provided by the invention has the activity of nitrogen-fixing enzyme under the conditions of limited ammonium and high ammonium. Whereas the wild type strain was only at 0-5mM NH ₄ ⁺ Nitrogen fixing enzyme activity only exists in the range.

The raw materials or the reagents involved in the invention are all common commercial products, and the related operations are all routine operations in the field unless specified.

For example, the BamHI/HindIII restriction enzyme used in the present invention was purchased from New England Biolabs. Plasmid pHY300PLK was purchased from Biovector plasmid vector strain cell gene collection. Paenibacillus polymyxa WLY 78A substitute for Paenibacillus polymyxa available from Shanghai Ke Wei chemical technology Co., ltd was also offered by the university of agricultural university, china, task group Chen Sanfeng.

Paenibacillus polymyxa WLY78 is already described in the literature (Comparative genomic analysis of N ₂ -fixing and non-N ₂ Fixing Paenibacillus spp. organization, evolution and expression of the nitrogen fixation genes, jian-BoXie etc., plos genetics, DOI:10.1371/journ al. Pgen.1004231).

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

EXAMPLE 1 construction of Ald Gene overexpressed Paenibacillus azotoformans

1. Primer and vector

aldPF：5′-ATGGAAAAACGCTTTGCCCAATATTCATCAAAAAGTCGAGC-3′(SEQ ID NO:2)；

aldR：5′-TTATAACAGGAATTCCCGGGCTATTGTAAGGCTTTCGC-3′(SEQ ID NO:3)。

pHY300PLK: shuttle plasmid, tc ^R The cell protein antibody gene collection (NTCC collection of typical cultures) was purchased from the Biovector plasmid vector strain.

2. The primers aldPF and aldR were used to amplify a fragment 1329bp long by PCR using the Paenibacillus polymyxa WLY genome as template, which contains the ald promoter region and the ald gene.

Cutting the gel with a Tiangen gel recovery kit to recover the fragments; the plasmid pHY300PLK was digested with BamHI and HindIII, digested at 37℃for 3 hours, and recovered by column chromatography.

The purified fragment and the digested and purified pHY300PLK were assembled together using Gibson assembly master mix (New England Biolabs) to give a recombinant vector pHY78ald.

The recombinant vector pHY78ald is transformed into Paenibacillus polymyxa WLY competent cells by electric shock, strains with tetracycline resistance are obtained by screening, and homologous double-exchanged ald overexpression strain WT/ald is obtained by PCR verification and sequencing.

EXAMPLE 2 determination of the activity of the Nitrogen-fixing enzyme

The ald gene overexpression strain WT/ald and the wild strain (WT) are respectively cultured in basic culture media added with different ammonium concentrations, and the activity of the nitrogen fixation enzyme is measured after more than 10 hours of culture (acetylene reduction method).

Nitrogen fixation enzyme Activity (nmol C) ₂ H ₄ Per mg protein hr) is calculated as follows:

the formulation of the minimal medium is as follows: 10.4g Na/liter of liquid medium ₂ HPO ₄ ,3.4g KH ₂ PO ₄ ,26mg CaCl ₂ ·2H ₂ O,30mg MgSO ₄ ,0.3mg MnSO ₄ ,36mg Ferric citrate,7.6mg Na ₂ MoO ₄ ·2H ₂ O,10mg of para-aminobenzoic acid, 5. Mu.g of biotin and 4g of glucose.

Specific methods for determining the activity of the enzyme immobilized on nitrogen (acetylene reduction method) can be found in A minimal nitrogen fixation gene cluster from Paenibacillus sp.WLY78 enables expression of active nitrogenase in Escherichia coll.Living Wang etc., PLoS Genet, DOI:10.1371/journ al pgen.1003865.

The experimental results are shown in FIG. 1, in which the wild-type strain (WT) was grown at 30-400mM NH ₄ ⁺ Under the condition of almost no activity of the nitrogen fixation enzyme, while the ald gene overexpression strain WT/ald strain is in 30-400mM NH ₄ ⁺ Can fix nitrogen under the condition that the concentration is 100mM NH ₄ ⁺ The activity of the nitrogen fixation enzyme under the condition can reach the level under the condition without ammonium.

EXAMPLE 3 cultivation of Paenibacillus azotoformans

Culturing nitrogen-fixing bacillus WT/ald in culture medium (sucrose 36g/L, tryptone 5g/L, yeast powder 11g/L, mgSO) ₄ 0.51g/L，NaCl 3.5g/L，Na ₂ MoO ₄ g/L，FeSO ₄ g/L), shaking culture at 30deg.C for 36-48 hr to obtain viable bacteria concentration of 10 ⁸ Each/g.

EXAMPLE 4 overexpression of the ald Gene from Paenibacillus azoxydans in Paenibacillus polymyxa

1. Primer and vector

aldPF:5′-atggaaaaacgctttgcccaATATTCATCAAAAAGTCGAGC-3′

aldP27R:5′-cgatgatcatTTCATTCACCTCTCCTAAAAATAATG-3′

T27aldF:5′-ggtgaatgaaATGATCATCGGAGTACCTAAG-3′

T27aldR:5′-ttataacaggaattcccgggTTAGATCAGGGTAAACTCTTG-3′

pHY300PLK: shuttle plasmid, tc ^R The cell protein antibody gene collection (NTCC collection of typical cultures) was purchased from the Biovector plasmid vector strain.

2. The ald gene (SEQ ID NO: 6) from Paenibacillus sabinae T (GenBank: CP 004078) was overexpressed in Paenibacillus polymyxa WLY78 and the effect on the activity of the enzyme azotase was determined. PCR amplification is carried out by using primers T27aldF and T27aldR and P.sabinae T27 genome as a template to obtain an ald gene coding region with the length of 1134 bp; PCR amplification is carried out by using primers aldPF and aldP27R and P.polymyxa WLY78 genome as a template to obtain an ald promoter region with length of 198bp, and the two fragments are recombined in a linearized pHY300PLK vector to obtain a recombined vector pHY27ald. pHY27ald was transformed into P.polymyxa WLY78 to give the ald gene overexpressing mutant WT/27ald.

Wild Type (WT) and WT/27ald mutant strains were each cultured in a medium containing 0mM NH ₄ Minimal medium of Cl (ammonium free medium, -N) and 100mM NH ₄ The results of culturing in Cl minimal medium (high ammonium medium, +N) and measuring the activity of the enzyme for more than 10 hours (acetylene reduction method) show that the wild-type strain (WT) has no activity of the enzyme under high ammonium conditions, and the WT/27ald strain can fix nitrogen under high ammonium conditions, indicating that the ald gene from other Paenibacillus azotoformans can induce P.polymyxa WLY78 to perform high ammonium fixation. EXAMPLE 5 construction of GlnR binding site mutant Strain of the ald Gene promoter region of Paenibacillus polymyxa WLY78

1. Primer and vector

P1：5′-ACGATGCGTCCGGCGTAGAGTACTGTCCCTTCGATAACAATCGC-3′

P2：5′-TGTAAAGGATCCTAAAGACATATACATAAGGAGAATGCCGATG-3′

P3：5′-TCTTTAGGATCCTTTACATGACATATATTCATCAAAAAGTCGAGCC-3′

P4：5′-CGCAAAAGACATAATCGATACTATTGTAAGGCTTTCGC-3′

pRN5101: temperature sensitive shuttle plasmid, em ^R Purchased from the Biovector plasmid vector strain cell gene collection.

2. Using primers P1 and P2, and using Paenibacillus polymyxa WLY genome as template to make PCR amplification to obtain 984bp sequence; PCR (polymerase chain reaction) amplification is carried out by using primers P3 and P4 and Paenibacillus polymyxa WLY genome as a template to obtain a sequence with the length of 1353 bp; the two fragments were recovered by cutting with the Tiangen gel recovery kit.

The temperature-sensitive plasmid pRN5101 was digested with BamHI and HindIII to obtain an enzyme fragment.

The recovered and purified 2 fragments and the cleaved and purified pRN5101 were assembled together using Gibson assembly master mix (New England Biolabs) to give vector pMald.

The vector pMPald is transformed into Paenibacillus polymyxa WLY78 competent cells by electric shock, subcultured at 39 ℃, screened to obtain single-crossover strains with erythromycin resistance, subcultured again, screened from the single-crossover strains without erythromycin resistance, and verified by PCR and sequencing to obtain the homologous double-crossover strains MPald with the mutation of the ald promoter region GlnR binding site.

The results of culturing the wild-type MPald mutant in a minimal medium containing 0mM and 100mM ammonium chloride and measuring the activity of the nitrogen fixation enzyme are shown in FIG. 3, and the MPald mutant has the activity of the nitrogen fixation enzyme under the condition of high ammonium, which indicates that P.polymyxa WLY78 can be induced to perform high ammonium nitrogen fixation by regulating the expression of the ald gene.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. An ammonium-resistant nitrogen-fixing microorganism overexpressing an ald gene, characterized in that the ammonium-resistant nitrogen-fixing microorganism is obtained by overexpressing an ald gene from a Paenibacillus microorganism in a Paenibacillus (Paenibacillus) microorganism;

the pathway of overexpression is selected from the following 1) to 6), or an optional combination:

1) Enhanced by introducing a plasmid having the gene;

2) Enhanced by increasing the copy number of the gene on the chromosome;

3) Enhanced by altering the promoter sequence of said gene on the chromosome;

4) Enhanced by operably linking a strong promoter to the gene;

5) Enhanced by the introduction of enhancers;

6) Enhanced by the use of a gene or allele having a high activity encoding an alanine dehydrogenase.

2. The ammonium resistant nitrogen fixing microorganism of claim 1, wherein the ald gene is derived from paenibacillus polymyxa (Paenibacillus polymyxa), paenibacillus nitrogen fixing (Paenibacillus sabinae).

3. The ammonium resistant nitrogen fixing microorganism of claim 2, wherein the nucleotide sequence of the ald gene is:

i) A nucleotide sequence shown in SEQ ID NO. 1;

ii) the nucleotide sequence shown in SEQ ID NO. 1 is substituted, deleted and/or added with one or more nucleotides and expresses the same functional protein;

iii) A nucleotide sequence which hybridizes to the sequence shown in SEQ ID No. 1 and expresses the same functional protein under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or in a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution; or (b)

iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

4. The ammonium resistant nitrogen fixing microorganism according to claim 1, wherein TGTCAT in the transcription regulatory factor GlnR binding site of the ald gene promoter region is replaced with GGATCC.

5. Ammonium resistant nitrogen fixing microorganism according to claims 1-4, characterized in that the starting strain is paenibacillus polymyxa, preferably paenibacillus polymyxa WLY78.

6. The construction method of the ammonium-resistant nitrogen fixation microorganism over-expressing the ald gene is characterized by comprising the following steps:

(1) PCR amplification is carried out by using primers aldPF and aldR and Paenibacillus polymyxa WLY78 genome as a template to obtain a fragment with a length of 1329 bp; cutting the glue to recover fragments by using a Tiangen glue recovery kit;

(2) The plasmid pHY300PLK was digested with BamHI and HindIII to obtain an enzyme fragment;

(3) Assembling the recovered fragment in the step (1) and the enzyme-digested fragment in the step (2) through Gibson to obtain a recombinant vector pHY78ald;

(4) Transforming the recombinant vector pHY78ald into competent cells of the Paenibacillus polymyxa WLY78, and screening strains with tetracycline resistance to obtain the strain;

wherein, the nucleotide sequences of the primers aldPF and aldR are respectively shown in SEQ ID NO. 2 and SEQ ID NO. 3.

7. The method of claim 6, wherein the conversion of step (4) is a shock conversion.

Application of ald gene in inducing paenibacillus microorganism to fix nitrogen under high ammonium condition;

the application comprises: using genetic engineering means to over-express the ald gene by said microorganism;

the ald gene is derived from a microorganism of the genus Paenibacillus, preferably Paenibacillus polymyxa, paenibacillus azoxygenus, more preferably Paenibacillus polymyxa WLY78.

9. Use according to claim 8, characterized in that the starting strain is paenibacillus polymyxa, preferably paenibacillus polymyxa WLY78.

10. The use according to claim 8 or 9, wherein the high ammonium condition is NH in the environment of the microorganism ₄ ⁺ The concentration of (C) is 30-400 mM.