CN119306809B

CN119306809B - Application of porin PorA in improving the ammonia nitrogen utilization capacity of methanogenic bacteria

Info

Publication number: CN119306809B
Application number: CN202411367723.5A
Authority: CN
Inventors: 闫新; 张海丽
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2024-09-29
Filing date: 2024-09-29
Publication date: 2025-11-04
Anticipated expiration: 2044-09-29
Also published as: CN119306809A

Abstract

This invention discloses the application of porin PorA in improving the ammonia nitrogen utilization capacity of methanogenic bacteria. A porin PorA, with its amino acid sequence shown in SEQ ID NO.1, is described. The encoding gene porA for the porin PorA, with its nucleotide sequence shown in SEQ ID NO.2, is also described. The application of porin PorA in improving the ammonium nitrogen utilization capacity of *Methylotuvimicrobium buryatense* is explained. Inactivation of this porin enables the methanogenic bacterium *Methylotuvimicrobium buryatense* to grow using ammonium nitrogen, reducing the cost of fermenting and culturing this bacterium, which is of great significance for its industrial application.

Description

Application of porin PorA in improving ammonia nitrogen utilization capacity of methane-oxidizing bacteria

Technical field:

The invention belongs to the technical field of biology, and relates to a method for improving ammonia nitrogen utilization capacity of methane-oxidizing bacteria Methylotuvimicrobium buryatense and application thereof.

The background technology is as follows:

Methane-oxidizing bacteria can grow by taking methane as the only carbon source and energy source, and can be widely distributed in the environments such as wetlands, lakes, oceans and the like, and play an important role in the circulation of methane on the earth. Since the last century, the role of this class of microorganisms in methane bioconversion and methane pollution abatement has been valued. Methane bioconversion refers to the production of single cell proteins, degradable plastics, carotenoids, tetrahydropyrimidines and other products by converting methane using methane-oxidizing bacteria. The methane pollution elimination is to eliminate the methane discharged by point sources such as refuse landfill and sewage treatment system by using methane oxidizing bacteria, and reduce the methane quantity entering the atmosphere.

While there have been many commercial attempts at methane bioconversion and many engineering efforts at methane elimination, there is currently no sustained success in both. For this reason, economy is one of the important limiting factors. One way to increase economy is to reduce the cost of large-scale cultivation of methane-oxidizing bacteria. From the medium feedstock point of view, the form of the nitrogen source affects the fermentation costs. Since methane-oxidizing bacteria are generally sensitive to ammonium nitrogen, nitrate nitrogen is typically used as the nitrogen source for the bacteria. However, in terms of price, potassium nitrate is about 5 times higher than ammonium chloride. If ammonium nitrogen can be used to replace nitrate nitrogen, the culture cost of methane-oxidizing bacteria can be reduced.

Methylotuvimicrobium buryatense is a methane-oxidizing bacterium with industrial application value. The methane-oxidizing bacteria have high growth speed, are alkalophilic, anti-pollution and resistant to impurities in natural gas (PNAS, 2023, https:// doi.org/10.1073/pnas.2310046120). In recent years, this class of bacteria has been developed as an important model strain and metabolically engineered chassis host. However, this species is very sensitive to ammonium nitrogen and is usually only cultivated with nitrate nitrogen. If the bacteria can better utilize ammonium nitrogen, the large-scale culture cost is reduced, and the method has important significance for industrial application.

The invention provides a method for improving the ammonia nitrogen utilization capacity of methane-oxidizing bacteria Methylotuvimicrobium buryatense by inactivating porin and application thereof in fermentation culture.

The invention comprises the following steps:

the invention aims to solve the problem that methane-oxidizing bacteria Methylotuvimicrobium buryatense are sensitive to ammonium nitrogen, and provides a method for improving the utilization capacity of ammonium nitrogen by inactivating porin and application thereof.

In order to solve the technical problems, the invention discloses the following technical scheme:

In a first aspect, the invention discloses porin PorA affecting Methylotuvimicrobium buryatense ammonium nitrogen utilization capacity and a coding gene PorA thereof.

Porin PorA has an amino acid sequence shown in SEQ ID NO. 1.

The coding gene PorA of porin has a nucleotide sequence shown in SEQ ID NO. 2.

In a second aspect, the invention discloses application of the porin PorA in improving Methylotuvimicrobium buryatense ammonium nitrogen utilization capacity.

Wherein, the inactivation of porin can improve Methylotuvimicrobium buryatense ammonium nitrogen utilization capacity.

Wherein, the inactivation of porin PorA can be achieved by gene knockout, gene mutation or gene silencing of PorA.

The application of the porin PorA coding gene in improving Methylotuvimicrobium buryatense ammonium nitrogen utilization capacity can improve Methylotuvimicrobium buryatense ammonium nitrogen utilization capacity through gene knockout, gene mutation or gene silencing of the PorA.

Application of substances for realizing porin PorA inactivation by gene knockout, gene mutation or gene silencing in improving Methylotuvimicrobium buryatense ammonium nitrogen utilization capacity.

In summary, the invention provides a method for improving the ammonia nitrogen utilization capacity of methane-oxidizing bacteria Methylotuvimicrobium buryatense by inactivating porin and application thereof.

The beneficial effects are that:

The invention discloses a porin which influences the ammonium ion uptake efficiency of methane-oxidizing bacteria Methylotuvimicrobiumburyatense, and deactivation of the porin can enable the methane-oxidizing bacteria Methylotuvimicrobium buryatense to grow by utilizing ammonium nitrogen, so that the cost of fermenting and culturing the microorganism is reduced, and the porin has important significance for industrial application of the microorganism.

Description of the drawings:

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1 shows a method for screening a 5GB1C mutant with high ammonia nitrogen utilization capacity.

FIG. 2 growth ability test of strains WT, deltapora and Deltapora-Com with ammonium salt as nitrogen source.

Fig. 3 a 3D architecture simulation of PorA.

FIG. 4 strain Deltapora was tested for the growth capacity of fed-batch ammonium chloride in shake flasks.

FIG. 5 strain delta porA is tested for accumulation of deleterious products during the use of ammonia nitrogen.

The specific embodiment is as follows:

according to the invention, firstly, a methanotrophic bacterium Methylotuvimicrobium buryatense mutant capable of tolerating ammonium nitrogen is screened through transposon random insertion mutation, and then the function of porA gene porA is determined through gene knockout and anaplerosis experiments.

Example 1

As shown in FIG. 1, the transposon random insertion library of strain Methylotuvimicrobium buryatense GBC1 was constructed using pSC123 plasmid carrying the Himar1 mariner transposon according to the method of reference (AEM, 2013.Https:// doi: 10.1128/AEM.02478-13). Methylotuvimicrobium buryatense 5GBC1 is a model strain of methane-oxidizing bacteria, which is derived from the reference (AEM, 2015.Https:// doi: 10.1128/AEM.03795-14). Mutants were screened on NMS2 medium and NMS2N (NMS2+8 mM NH ₄ Cl) medium, respectively. The starting strain can only grow on NMS2 plates and cannot grow on NMS2N plates. A mutant capable of growing on NMS2N was selected by this method and designated 5G-P. The position of transposon insertion in mutant 5G-P was determined by the chromosome walking method by the method of the reference (AEM, 2007, https:// doi. Org/10.1128/AEM.02973-06), and the gene which was inserted and inactivated was designated porA. Note that mutant 5G-P can be obtained by genetic engineering means or repeatedly by the method of "example 2".

Example 2

To verify that inactivation of porA would increase the ability of strain 5GBC1 to utilize ammonium nitrogen, strain Δpora was obtained by knockout of porA on the basis of strain 5GBC1 by a marker-free knockout system based on the pheS ^AG gene (Frontiers in Microbiology,2020, https:// doi.org/10.3389/fmib.2020.00441). Specific steps were as follows, using a fragment of about 450bp downstream of the target region to be deleted as a forward repeat (DIRECT REPEAT, DR) sequence and adding to the PZ cassette (PZ cassette contains tac promoter, RBSmmoX, artificially synthesized pheS ^AG and zeo resistance gene). Then, the left and right homology arms (LF, RF) were fused in the order of LF-DR-PZ-RF by overlap PCR, and the fused DNA fragments were purified and recovered and transferred into competent cells of 5GB1C by electrotransformation (Frontiers in Microbiology,2020, https:// do i.org/10.3389/fmib.2020.00441). And (3) screening the transformants successfully recombined into the target region through bleomycin expression cassettes DR and PZ, then carrying out reverse screening on the transformants obtained by the first step of screening through p-Cl-Phe, and obtaining the traceless knockout strain through PCR identification. Wherein the fragment LF is amplified by the primer LF-F (GCAGAAGAAGAACGGCAAAT) and the primer LF-R (AAAAGGC GACACTTGGTTTTGAAAACGGCCTCAAA), the fragment DR is amplified by the primer DR-F (AGGCCGTTTTCAAAACCAAG TGTCGCCTTTTTAAGTA) and the primer DR-R (TGTCAACAGCTCATTTCAGAGTGAGTATAAAGGGAGTATGTCG), the fragment PZ is amplified by the primer PZ-F (CTCTGAAATGAGCTGTTGACA) and the primer PZ-R (TCAGTCCTGCTCCTCGGCCA C), and the fragment RF is amplified by the primer RF-F (GTGGCCGAGGAGCAGGACTGACTAGAAACGAATGATCGCATC) and the primer RF-R (TACGGCGTGGATGCTGCTTAT).

The low transcription site (160485) in the genome of 5GB1C is used as an integration site for the porA complementation, and the porA is further integrated on the chromosome of the Δpora by a homologous double exchange method to obtain a replenisher strain Δpora-Com. The method comprises the specific steps of amplifying an upstream and downstream homology arm CLF and CRF of a reconnaissance site and a porA gene fragment CPA containing an original promoter region by using a strain 5GB1C total DNA as a template, amplifying a kanamycin resistance gene fragment KAN (AEM, 2015, http:// dx.doi.org/10.1128) by using pAWP as a template, fusing the four fragments according to the sequence of CLF, CPA, KAN, CRF, and transferring the recovered fragments into competent cells of the strain delta porA in an electrotransformation mode. The target strain was screened by kanamycin, and PCR and sequencing verified. Wherein the fragment CLF is amplified by the primer CLF-F (GGTGTCGATGGCATGCTCAA) and the primer CLF-R (CA AGGCGAAGTTGAAGGCGC), the fragment CPA is amplified by the primer CPA-F (GCGCCTTCAACTTCGCCTTGACCGTCGT GAACCCATTCAT) and the primer CPA-R (TGCTCGATGAGTTTTTCTAACTAGAAACGAATGATCGCAT), the fragment KAN is amplified by the primer KAN-F (TTAGAAAAACTCATCGAGCA) and the primer KAN-R (CGCGTATAGCTTGCCGGAAG), and the fragment CRF is amplified by the primer CRF-F (CTTCCGGCAAGCTATACGCGATCGATCTCCGCGATAATCT) and the primer CRF-R (GTTACAGGCGTTACGTTACGTT).

As shown in FIG. 2, strain Δpora can be grown in AMS2 medium (other components in the medium except nitrogen source change are the same as NMS2 medium, ammonium chloride is used as nitrogen source) with 1mM and 2mM NH ₄ Cl, while starting strain 5GBC1 and anaplerotic Δpora-Com are difficult to grow. These results demonstrate that inactivation of porA can increase the ability of strain 5GBC1 to utilize ammonium nitrogen.

Structural simulations of PorA were performed by AlphaFold, and as shown in fig. 3, the results showed that PorA has the typical characteristics of porins, consisting of 16 β chains arranged in β -barrels, organized as trimers. The interior of the channel appears hourglass-shaped, with the narrowest part being the "constriction", and the outer ring, as seen in a cross-section perpendicular to the structure.

Example 3 inactivation of porA Gene encoding porA increase Methylotuvimicrobium buryatense GBC1 Using ammonium Nitrogen

To verify whether the porA knockout strain Δpora could achieve higher biomass by using ammonium nitrogen, the strain Δpora was grown overnight in AMS2 medium at 30 ℃ at 180rpm in shake flasks, then 1% inoculated into 50ml of AMS2 broth at 30 ℃ at 180rpm, supplemented with 0.5mM NH ₄ Cl every 6 hours, and the gas in shake flasks was replaced every 12 hours. As shown in FIG. 4, the strain Δpora can continuously grow under the condition of continuously supplementing NH ₄ Cl, which shows that ammonium nitrogen can be utilized to replace nitrate nitrogen for fermenting and culturing the strain Δpora, and the method has important significance in reducing the cost of Methylotuvimicrobiumburyatense fermenting and culturing.

Example 4 inactivation of porA Gene encoding porA reduces detrimental product accumulation

The methane-oxidizing bacteria can convert ammonia into harmful substances such as hydroxylamine, nitrite and nitrous oxide, and in order to detect whether the harmful substances are reduced after the inactivation of the gene porA, a starting strain Methylotuvimicrobium buryatense GBC1 and a knockout strain Deltapora are respectively placed in NMS2 culture and cultured at 30 ℃ and 180rpm overnight to reach an exponential phase, then bacteria are centrifugally collected, transferred to AMS2 culture medium and cultured at 30 ℃ and 180rpm overnight, and the content of accumulated hydroxylamine, nitrite and nitrous oxide is detected (see methods ANALYTICAL CHEMISTRY,1955, https:// doi.org/10.1021/ac60106a054 and PNAS,2016, https:// doi.org/10.1073/pnas.1611113). As shown in FIG. 5, the strain Δpora had about 60% lower nitrite accumulation in the culture medium supernatant, about 50% lower hydroxylamine accumulation and about 50% lower nitrous oxide accumulation than the starting strain.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The application of porin PorA in improving the ammonium nitrogen utilization capacity of methane-oxidizing bacteria Methylotuvimicrobium buryatense is that the porin PorA is deactivated to improve the Methylotuvimicrobium buryatense ammonium nitrogen utilization capacity, and the amino acid sequence of the porin PorA is shown as SEQ ID NO. 1.

2. The application of the porin PorA encoding gene PorA in improving the ammonium nitrogen utilization capacity of methane-oxidizing bacteria Methylotuvimicrobium buryatense, which is characterized in that the Methylotuvimicrobium buryatense ammonium nitrogen utilization capacity is improved by gene knockout or gene silencing of the PorA, and the nucleotide sequence of the PorA encoding gene PorA is shown as SEQ ID No. 2.