CN116004494B - Method for improving growth and colonisation of alpha-proteobacteria phylum methyl nutrition bacteria in plant phyllosphere - Google Patents
Method for improving growth and colonisation of alpha-proteobacteria phylum methyl nutrition bacteria in plant phyllosphere Download PDFInfo
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Abstract
The application discloses a method for improving growth and colonization of alpha-proteobacteria phylum methyl nutrition bacteria in plant phyllospheres. The method is characterized in that alpha-proteobacteria phylum methyl nutrition bacteria are improved, and the mutant strain of the alpha-proteobacteria phylum methyl nutrition bacteria or the modified strain of the alpha-proteobacteria phylum methyl nutrition bacteria or one or more combinations of engineering bacteria of the alpha-proteobacteria phylum methyl nutrition bacteria are improved; then, the mutant strain of the alpha-proteobacteria phylum methyl nutrition bacteria or the modified strain of the alpha-proteobacteria phylum methyl nutrition bacteria or one or more combinations of engineering bacteria of the alpha-proteobacteria phylum methyl nutrition bacteria are colonized on leaf boundaries of plants of different families. The application can effectively improve the application capability of alpha-proteobacteria phylum methyl nutrition bacteria as plant probiotics and phyllospheric bacterial fertilizer.
Description
Technical Field
The application relates to a method for effectively improving growth and colonization of plant phyllosphere growth of alpha-proteobacteria phylum methyl nutrition bacteria under the condition of an oligocarbon source, belonging to the fields of microbiological technology and modern agriculture.
Background
Methyl trophic bacteria (methyl trophs) are capable of utilizing methanol, a byproduct released by metabolism of pectin on plant phyllospheres, as a carbon source and energy source for growth, and are a phyllosphere dominant flora of many plants and crops (such as arabidopsis thaliana, wheat, soybean, corn, rice, alfalfa, tobacco, tomatoes, melons, peppers and cabbages). Methylotrophic bacteria (e.g. methylobacterium oryzae)Methylorubrum oryzae、Methylobacillus nodulationM. nodulans、Methylobacillus torvus (L.) RickenM. extorquensMethylobacillus radioduransM. radiotolerans、Methylobacillus floccusM. gregansAnd Methylobacillus spanishM. hispanicum) Can release auxin and mitogen to promote plant growth, can release extracellular enzyme and ferritin to convert phytic acid into soluble phosphorus, promote iron absorption or have biological nitrogen fixation capacity, and can synthesize secondary metabolic substances for inhibiting the growth of pathogenic microorganisms to play a role in biological control. Therefore, the methyl nutrition bacteria have the advantages of remarkably improving the growth and production capacity of crops in natural environment and adverse conditions such as drought, salt and alkali, diseases and the like, reducing the dependence on organic fertilizers and chemical pesticides, promoting the stable and high yield of crops, and are a plant probiotics with great development and application prospects. However, the plant phyllosphere environment is complex, and particularly compared with the plant rhizosphere, the phyllosphere faces the adversity of nutrient element scarcity. Research reports that the quantity of methanol released by crop leaves is obvious along with fluctuation of crop types, surrounding environment factors and the like, the quantity of methanol released per gram of dry weight leaves per hour is unequal from 0.5 mug to 35.0 mug, and the release rate is less than 1.0 mug g -1 h -1 And the concentration of other carbon sources is also trace.
Therefore, research on how to strengthen the growth capacity of the methylotrophic bacteria in the oligotrophic phyllosphere environment can obviously promote the phyllosphere colonization effect of the methylotrophic bacteria and improve the growth and production of crops.
Disclosure of Invention
The application aims to provide a method for improving the growth and colonization of alpha-proteobacteria phylum methyl nutrition bacteria in plant phyllospheres, so as to make up the defects of the prior art.
Phosphoribosyl pyrophosphate kinase (Ribose-phosphate diphosphokinase), the first rate-limiting enzyme for de novo synthesis of purine nucleotides, pyrimidine nucleotides, histidine, tryptophan, and reducing power, catalyzes the phosphorylation of Ribose-5-phosphate (Ribose-5-phosphate) to phosphoribosyl pyrophosphate (Phosphoribosyl pyrophosphate). The de novo synthesis of purine nucleotides, pyrimidine nucleotides, etc., consumes large amounts of ATP and precursor metabolites. The Phosphoketolase pathway (Phosphoketolase pathway) is capable of catalyzing the cleavage of Fructose-6-phosphate (fr-6-phosphate) to Erythrose-4-phosphate (Erythrose-4-phosphate) and Acetyl phosphate (Acetyl-phosphate) or Xylulose-5-phosphate (Xylulose-5-phosphate) to Glyceraldehyde-3-phosphate and Acetyl phosphate using Phosphoketolase (phosphokinase). The acetyl phosphate is then converted to the important central metabolite acetyl-coa. The phosphoketolase pathway does not produce carbon dioxide during the synthesis of acetyl-coa, and is carbon neutral. alpha-Proteus methylotrophic bacteria (e.g. M.species, M.torpedoM. extorquens) The Serine cycle (Serine cycle) coupled Ethylmalonyl-CoA pathway was used to assimilate methanol and carbon dioxide, i.e. to assimilate 2 molecules of methanol and 4 molecules of carbon dioxide to synthesize 1 molecule of acetyl-CoA and 2 molecules of glyoxylic acid, but blocking the phosphoketolase pathway did not affect normal cell growth, indicating that this pathway was efficient under standard laboratory culture conditions (i.e.: methanol concentration 120 mM) is an optional metabolic pathway. Previous reports also indicate that when alpha-proteobacteria are colonized on plant leaves, other multi-carbon substances (such as oxalic acid, pyruvic acid and succinic acid) can be utilized as carbon sources besides methanol as a main carbon source.
In order to effectively improve the colonization and growth of alpha-proteobacteria phylum-methyl vegetative bacteria on plant leaves, a technical strategy is needed to improve the growth and colonization capacity on plant leaves by optimizing the metabolic flux distribution of central metabolism so that the cell metabolism under an oligocarbon source is more balanced and efficient. The research of the application shows that the function of phosphoribosyl pyrophosphate kinase is regulated down in alpha-proteobacteria phylum-methyl nutrition bacteria, or the metabolic flux of phosphoketolase pathway is improved, the growth capacity of alpha-proteobacteria phylum-methyl nutrition bacteria on an oligocarbon source can be obviously enhanced, and the plant phyllosphere colonization effect is obviously better than that of wild bacteria.
In combination with the above research, in order to achieve the above purpose, the specific technical scheme adopted by the application is as follows:
a mutant strain of alpha-proteobacteria phylum methyl nutrition bacteria is based on the alpha-proteobacteria phylum methyl nutrition bacteria, and the phosphoribosyl pyrophosphate kinase of the alpha-proteobacteria phylum methyl nutrition bacteria is mutated by a protein amino acid sequence mutation technology, a nucleotide sequence mutation technology or other mutation technologies so as to reduce phosphoribosyl pyrophosphate kinase catalytic activity.
An altered strain of alpha-proteobacteria phylum methyl trophic bacteria, which is based on alpha-proteobacteria phylum methyl trophic bacteria, and reduces the transcription level or translation level of phosphoribosyl pyrophosphate kinase genes by modern molecular biology technology; such modern molecular biology techniques include, but are not limited to, CRISPRi techniques, RNAi techniques, gene promoter or ribosome binding site sequence substitution techniques and the like.
The engineering bacteria of alpha-proteobacteria phylum methyl nutrition bacteria are based on the alpha-proteobacteria phylum methyl nutrition bacteria, and can increase the metabolic flux of the phosphoketolase metabolic pathway by using a plasmid gene overexpression technology, or by increasing the copy number of a target gene on the genome of the alpha-proteobacteria phylum methyl nutrition bacteria, or by using a genome gene element replacement modification technology, or by adopting other molecular biology technologies, and improving the expression level of genes related to the phosphoketolase metabolic pathway of the alpha-proteobacteria phylum methyl nutrition bacteria.
Further, the alpha-proteobacteria phylum methyl nutrient bacteria is prepared from organic carbon one including methane, methanol and formaldehydeBacterial colonies of the type having pink color, including but not limited to Methylobacillus oryzae, are coupled by serine circulatory pathway to ethylmalonyl-CoA pathway and tricarboxylic acid circulatory assimilation of organic carbonM. oryzae、Methylobacillus nodulationM. nodulans、Methylobacillus torvus (L.) RickenM. extorquensMethylobacillus radioduransM. radiotolerans、Methylobacillus floccusM. gregansAnd Methylobacillus spanishM. hispanicum。
Furthermore, the gene related to the metabolic pathway of the phosphoketolase is phosphoketolasexfp) Phosphoric acid transacetylasepta) Acetic acid kinase [ ]ack) acetyl-CoA synthetase ]acs) Etc.
The application of the mutant strain of the alpha-proteobacteria phylum methyl nutrition bacteria or the modified strain of the alpha-proteobacteria phylum methyl nutrition bacteria or one or more combinations of engineering bacteria of the alpha-proteobacteria phylum methyl nutrition bacteria in plant growth.
Further, the mutant strain of the alpha-proteobacteria phylum methyl nutrition bacteria or the modified strain of the alpha-proteobacteria phylum methyl nutrition bacteria or the application of one or more combinations of engineering bacteria of the alpha-proteobacteria phylum methyl nutrition bacteria in improving the growth capacity of the alpha-proteobacteria phylum methyl nutrition bacteria on an oligo carbon source or improving the plant phyllospheric growth colonisation.
Further, the application of the mutant strain of the alpha-proteobacteria phylum methyl nutrition bacteria or the modified strain of the alpha-proteobacteria phylum methyl nutrition bacteria or one or more combinations of engineering bacteria of the alpha-proteobacteria phylum methyl nutrition bacteria in preparing plant probiotics or phyllospheric bacterial manure.
Further, the mutant strain of the alpha-proteobacteria phylum-methyl nutrition bacteria or the modified strain of the alpha-proteobacteria phylum-methyl nutrition bacteria or the engineering bacteria of the alpha-proteobacteria phylum-methyl nutrition bacteria have the growth capacity which is obviously higher than that of wild bacteria on different oligocarbon sources (such as methanol, ethanol, acetic acid, oxalic acid, pyruvic acid and succinic acid), including shortening the growth delay period, improving the growth rate and improving the biomass conversion rate.
A method for improving growth and colonization of alpha-proteobacteria in plant phyllosphere, the method is firstly improved aiming at alpha-proteobacteria, namely, one or more of mutant strains of alpha-proteobacteria, modified strains of alpha-proteobacteria or engineering bacteria of alpha-proteobacteria; then, the mutant strain of the alpha-proteobacteria phylum methyl nutrition bacteria or the modified strain of the alpha-proteobacteria phylum methyl nutrition bacteria or one or more combinations of engineering bacteria of the alpha-proteobacteria phylum methyl nutrition bacteria are colonized on leaf boundaries of plants of different families.
Further, the method specifically comprises the following steps: liquid culturing one or more of the mutant strain of the alpha-proteobacteria phylum-methyl nutrition bacteria or the modified strain of the alpha-proteobacteria phylum-methyl nutrition bacteria or the engineering bacteria of the alpha-proteobacteria phylum-methyl nutrition bacteria to the middle and late period of the growth index, and adjusting the concentration of the bacteria to OD 600 About 0.5, soaking plant seeds or drip irrigation plant roots or spraying plant phyllospheres by using the bacterial suspension, and applying the bacterial agent for 3 weeks, wherein the colonization amount of the plant She Ji-Proteus methyl nutrition bacteria reaches 10 per gram of phyllosphere plant fresh weight 8 -10 9 CFU or more.
Further, the plant phyllosphere refers to a part of a plant which appears above the ground surface and comprises flowers, stems, leaves and fruits; such plants include, but are not limited to, vegetables and fruit crops such as wheat, cabbage, melon, tomato, water spinach, and other plants that are colonized by the alpha-proteobacteria phylum methyl nutrition She Jiding.
Further, the low carbon source, for example, low concentration methanol, is 15% lower than the laboratory standard culture methanol (120 mM), i.e., less than 18 mM, and the other low carbon source is the same equivalent carbon concentration as methanol.
The application has the advantages and beneficial effects that:
the application specifically provides improved strains of three alpha-proteobacteria phylum-methyl nutritional bacteria, which can down regulate the functions of phosphoribosyl pyrophosphate kinase or improve the metabolic flux of phosphoketolase metabolic pathways, can improve acetyl coenzyme A synthesis, so that the central metabolism under an oligocarbon source is more effectively balanced, the growth capacity of the alpha-proteobacteria phylum-methyl nutritional bacteria on the oligocarbon source is obviously improved, and finally the colonization effect on the phyllospheres of plants of different families is obviously improved.
The application provides a method for enabling alpha-proteobacteria to grow and colonize on an oligocarbon source and plant phyllospheres more effectively, and can effectively improve the application capability of the alpha-proteobacteria as plant probiotics and phyllospheres bacterial manure. Practice proves that the application obtains a technical strategy for effectively improving the growth capacity of alpha-proteobacteria phylum-methyl nutrition bacteria on an oligocarbon source, has obvious effect of improving plant phyllostachys colonization, and has good application potential for promoting crop growth and production.
Drawings
FIG. 1, wherein (a) is Methylobacillus cereusM. extorquensAM 1) three-dimensional structure diagram of phosphoribosyl pyrophosphate kinase, and (b) in vitro enzyme activity measurement result diagram.
FIG. 2 is a graph showing the results of decreasing phosphoribosyl pyrophosphate kinase activity and increasing the growth capacity of methylobacterium wraparound at the concentration of oligomethanol; wherein WT:M. extorquenswild strain of AM 1; WT-prs EVO ：M. extorquensMutant strain of phosphoribosyl pyrophosphate kinase of AM 1.
FIG. 3 is a graph showing the results of decreasing phosphoribosyl pyrophosphate kinase activity and increasing the growth capacity of M.torpedo oligo succinic acid (a, b) and oxalic acid (c, d) at the concentration, wherein WT:M. extorquenswild strain of AM 1; WT-prs EVO ：M. extorquensMutant strain of phosphoribosyl pyrophosphate kinase of AM 1.
FIG. 4 is a graph showing the results of CRISPRi interfering technology in decreasing transcription level of phosphoribosyl pyrophosphate kinase gene and increasing growth capacity of M.torpedo and M.oryzae at oligomethanol concentration, wherein WT12 and WT13:M. extorquenstargeting in AM1prsStrains with different nucleotide sites (275, 390) of the gene; WT04: a control strain; OR03 and OR04:M. oryzeamiddle targetingprsStrains with different nucleotide sites (274, 390) of the gene; OR01: control strain.
FIG. 5 shows that the promoter replacement technique reduces transcription of phosphoribosyl pyrophosphate kinase geneResults of increased level of growth capacity at concentration of methylobacterium torvum oligomethanol, wherein WT:M. extorquensAM1 wild strain; WT03: promoter replacement technology reductionM. extorquensAM1 inprsEngineered strains at the level of gene transcription.
FIG. 6 is a graph showing the results of mutant upregulation of phosphoketolase in M.torvum by phosphoribosyl pyrophosphate kinase.
FIG. 7 is a diagram showing overexpression of phosphoketolase pathway-related genesxfpResults for increasing growth capacity at concentration of methylobacterium torvum oligos, wherein WT10: comprising an over-expressed empty plasmid pCM80M. extorquensAM1 strain; WT11: overexpression using pCM80xfpGene of geneM. extorquensAM1 engineering strain.
FIG. 8 shows the increase of genes involved in the pathway of genomic phosphoketolasexfpResults of copy number increase growth ability at concentration of methylobacterium torvum oligomethanol, wherein WT:M. extorquensAM1 wild strain; WT02: genome augmentationxfpCopy number of geneM. extorquensAM1 engineering strain.
FIG. 9 is a graph comparing results of mutant strain, engineered strain and engineered strain of M.torpedo phosphoribosyl pyrophosphate kinase having significantly higher ability to colonize Arabidopsis thaliana She Jiding than the wild strain, wherein WT:M. extorquensAM1 wild strain; WT-prs EVO ：M. extorquensA mutant phosphoribosyl pyrophosphate kinase strain of AM 1; WT02: genome augmentationxfpCopy number of geneM. extorquensAn AM1 engineered strain; WT03: promoter replacement technology reductionprsAt the level of gene transcriptionM. extorquensAM1 engineered strain.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be further described with reference to examples, which are only a part of examples, but not all examples, of the present application, and the present application is not limited by the examples described below.
The application cultures the alpha-proteobacteria phylum methyl nutrition bacteria under 30 ℃ in laboratory culture conditions; by a means ofThe culture mode is preferably shake flask culture with the rotation speed of 200 rpm, and in the application, the preparation method of the Hypho culture medium is preferably as follows: macroelement (2X): macroelement a:5.06 g/L K 2 HPO 4 ，2.585 g/L NaH 2 PO 4 The method comprises the steps of carrying out a first treatment on the surface of the Macroelement B:0.4095 g/L MgSO 4 ·7H 2 O，1 g/L (NH4) 2 SO 4 The method comprises the steps of carrying out a first treatment on the surface of the If a solid medium is prepared, agar 30 g/L is added to macroelement B. Separately sterilizing, and using according to 1: mixing the materials according to the proportion of 1. Trace elements: the trace element A and the trace element B are stored separately, and 100 mL 1000X trace elements are prepared. Trace element A:1 g Na 2 EDTA，0.1 g FeSO 4 ·7H 2 O, adjusting the pH to 4 by using 1M NaOH; trace element B:0.14 g CaCl 2 ·2H 2 O，0.1 g MnCl 2 ·4H 2 O，0.02 g Na 2 MoO 4 ·2H 2 O，0.03 g CuSO 4 ·5H 2 O，0.16 g CoCl 2 ·6H 2 O，0.44 g ZnSO 4 ·7H 2 O, adjust pH to 1-2 with HCl. Filtering and sterilizing with 0.22 μm sterile filter after microelement preparation, sterilizing under non-autoclaving, refrigerating at 4deg.C, and diluting by 1000 times. When the method is used, a proper amount of carbon source is added in proportion, the concentration of methanol in the laboratory standard culture condition is 120 mM, the concentration of the oligoconcentration methanol is lower than 15% of the concentration of the methanol in the laboratory standard culture condition (120 mM), namely lower than 18 mM, the concentration of other low-concentration carbon sources is equal to the equivalent concentration of carbon in the methanol, in the specific embodiment of the application, 5mM, 10 mM and 15 mM of methanol concentration are selected, 1 mM and 3 mM of oligoconcentration succinic acid are selected, and 1 mM and 5mM of oligooxalic acid are selected. Specific examples were studied on behalf of methylobacterium wraparound.
Example 1 reduction of phosphoribosyl pyrophosphate kinase Activity by site-directed mutagenesis
The present example provides a method for reducing in vitro enzyme activity of phosphoribosyl pyrophosphate kinase (PRS, sequence as shown in SEQ 1) from methylobacterium torvum by site-directed mutagenesis, comprising the following steps:
this example uses the Swiss-model in-line tool to model homology to the target protein, with the template selected as the sourceBurkholderia pseudomalleiThe complex structure phosphoribosyl pyrophosphate kinase (PDB ID: 3 DAH) containing the ligand has the sequence consistency of more than 57 percent with the phosphoribosyl pyrophosphate kinase of the methylobacterium torvum and establishes a three-dimensional structure model of the target protein. The modeled complex structure was visually analyzed and processed using Pymol software. The results show that the D38 site of phosphoribosyl pyrophosphate kinase directly binds to the substrate ATP via hydrogen bonds, and that when D38 is mutated to N38, the binding capacity to ATP is significantly weaker, mainly because the oxygen atom of the D38 side chain has a stronger electronegativity, the end of the N38 side chain is one oxygen atom by one nitrogen atom, wherein the oxygen atom is similar to D38, and the nitrogen atom and NH of the base in the ATP molecule 2 The groups are difficult to form hydrogen bonding (fig. 1 a).
Gene encoding phosphoribosyl pyrophosphate kinase by overlap extension PCRprsThe mutation of G112 of (META1_4249) to A112, i.e.the mutation of the amino acid residue D38 of the enzyme to N38, the construction of the wild-type sequence and the mutant sequence, respectively, in the overexpressing plasmid pET32, is transformed intoE. coliIs used for over-expression to obtain the target protein. The transformant obtained was cultured at 200 rpm and 37℃and the OD was set 600 When the expression of target protein is=0.4-0.6, IPTG is added to induce the expression of target protein. At OD 600 When=1.7, cells were collected. 200 ml phosphate buffer (50 mM KH) 2 PO 4 5.5 mM imidazole, 150 mM NaCl,0.25 mM DTT,pH =7.9), cells were disrupted by a French Press cell disrupter, and the cell disruption solution was 1.6x10 4 g, centrifuging at 4 ℃ for 30 min, and purifying the recombinant wild type PRS and the mutant PRS respectively to the purity of more than 90% by utilizing nickel column affinity chromatography on the supernatant.
The enzyme activity of the wild type and mutant type of the phosphoribosyl pyrophosphate kinase PRS is detected through three-step cascade reaction, and the specific process is as follows:
①、Ri5P + ATP + PRS = PRPP + AMP
②、AMP + ATP + MK = 2 ADP
③、2 ADP + 2 PEP + LDH = 2 pyr + 2 ATP
④、2 Pyruvate + 2 NADH +2H + =2 Lactate + 2 NAD + +2H
by enzyme-linked reaction, with the addition of 5mM 5-phosphoribosyl-phosphate (Ri 5P), 5mM ATP,1 mM NADH,2 mM phosphoenolpyruvate, with the addition of 10 ug phosphoribosyl-pyrophosphate kinase PRS wild-type and mutant pure enzyme proteins, 2U myokinase, 2U pyruvate kinase, 2U lactate dehydrogenase, in a formulated Buffer (50 mM KH 2 PO 4 Ph=7.9, 150 mM NaCl,0.25 mM DTT), at 30 ℃, phosphoribosyl pyrophosphate kinase is added for 15 min and consumption of NADH is detected by uv spectrophotometer at 340 nm to characterize phosphoribosyl pyrophosphate kinase activity. Respectively, in OD with a system without phosphoribosyl pyrophosphate kinase and without substrate as a control 340 And detecting the consumption of the NADH, and calculating the consumption rate of the NADH by combining the extinction coefficient of the NADH to characterize the enzyme activity of the phosphopyrophosphoric acid synthetase. The enzyme activity analysis revealed that the mutant phosphoribosyl pyrophosphate kinase had about 42% lower than the enzyme activity (FIG. 1 b).
EXAMPLE 2 phosphoribosyl pyrophosphate kinase mutation to enhance the growth ability of M.toxygenum under oligocarbon Source conditions
In the embodiment, the mode strain methylobacterium wraparound of the alpha-proteobacteria phylum methyl nutrition bacteria is obtained by a homologous recombination methodM. extorquens) Mutant phosphoribosyl pyrophosphate kinase strain, and gene encoding phosphoribosyl pyrophosphate kinaseprsThe mutation of nucleotide 112 of (META1_4249) from G to A results in the mutation of the encoded amino acid from aspartic acid D to asparagine N. According to the difference of the different carbon sources entering the center metabolism of the methylobacterium wrenchii, the growth capacities of mutant strains and wild strains are analyzed by taking methanol, succinic acid and oxalic acid as representative carbon sources. The growth phenotype is determined by first culturing the strain in an oligomethanolic carbon source (e.g., 5mM, 10 mM,15 mM). It was found that a mutant strain of M.sprain phosphoribosyl pyrophosphate kinase (WT-prs EVO ) The delay time is two and a half hours shorter than that of the wild strain (WT) under the condition of low methanol concentration, and the maximum biomass is obviously higher than that of the wild strain, and the mutant strain (WT) of the phosphoribosyl pyrophosphate kinase of the methylobacterium is torqued under the condition of 5mM, 10 mM and 15 mM methanolprs EVO ) Maximum biomass fraction of (2)26.7%, 13.2% and 7.0% higher than the wild strain (WT) (FIGS. 2 a-d), while the mutant strain of M.wraparound phosphoribosyl pyrophosphate kinase (WT) was determined using the ab241033 methanol assay kitprs EVO ) The methanol consumption rate at 10 mM methanol was found to be 9.953.+ -. 0.133 mmol/g DCW/h, which was not significantly different from the wild-type (WT) methanol consumption rate (9.904.+ -. 0.111 mmol/g DCW/h) (FIG. 2 e), whereas the mutant strain of M.methylotrophicus phosphoribosyl pyrophosphate kinase (WT)prs EVO ) The biomass yield was 0.299.+ -. 0.002 g DCW/g methanol, which was significantly higher than that of the wild-type (WT) strain (0.286.+ -. 0.002 g DCW/g methanol) (FIG. 2 f), demonstrating that phosphoribosyl pyrophosphate kinase mutation significantly improved methanol utilization and growth capacity of M.torpedo under low concentration methanol carbon source. Mutant strain of M.torpedo phosphoribosyl pyrophosphate kinase (WT-prs EVO ) The growth ability of the mutant strain (WT) was improved to some extent under both of the oligosaccharide concentration succinic acid and oxalic acid carbon source, and the growth ability was improved under the culture conditions of 1 mM and 3 mM succinic acidprs EVO ) The lag phase of (a) was shortened by 10.7% and 13.3% respectively, compared to the wild strain (WT) (fig. 3a, b). Under conditions of 1 mM and 5mM oxalic acid, the mutant strain (WT-prs EVO ) The maximum biomass was increased by 33.3% and 13.2%, respectively (fig. 3c, d).
Example 3 reduction of transcription level of the phosphoribosyl pyrophosphate kinase Gene of alpha-Proteobacteria to increase growth ability in the presence of an oligoconcentration of methanol carbon source
In the embodiment, the CRISPRi interference technology is utilized to reduce the transcription level of the mode strain, namely the methylobacterium sprain and the methylobacterium oryzae phosphoribosyl pyrophosphatase gene of the alpha-proteobacteria, so that the growth capacity of the two strains under the condition of low concentration methanol is improved. Three different nucleotide sites of the phosphoribosyl pyrophosphate kinase gene are respectively targeted. The obtained interference strains were cultured under the condition of low concentration methanol (5 mM,10 mM,15 mM) respectively, and the growth phenotype was determined. The interfering strain (WT 12) of the methylobacterium torvum targeting nucleotide 275 of the phosphoribosyl pyrophosphate kinase gene has significant growth advantages under the condition of low concentration methanol compared with the control strain (WT 04), the delay period is shortened by three hours compared with the control strain, and the maximum biomass is respectively improved by 12.3 percent, 8.4 percent and 7.7 percent under the condition of 5mM, 10 mM and 15 mM methanol (figures 4 a-c); the rice methylobacterium targeted with different nucleotide sites of the phosphoribosyl pyrophosphate kinase gene has obvious growth advantages compared with a control strain under the condition of low concentration methanol, wherein the delay period of the targeted 274 nucleotide site interfering strain (OR 03) is shortened by more than six hours under the condition of 5mM and 10 mM methanol compared with that of the control strain (OR 01), and the maximum biomass is improved (figures 4d and e), which shows that the proper reduction of the transcription level of the phosphoribosyl pyrophosphate kinase gene under the condition of low concentration methanol can effectively improve the growth capacity of alpha-proteobacteria such as methylobacterium torvum, rice methylobacterium and the like.
By using a promoter replacement technology, the mode strain of the alpha-proteobacteria phylum methyl nutrition bacteria is subjected to demethylabacterium writhing by a homologous double exchange methodprsReplacement of the gene promoter with weaker strength in methylobacterium torvumgnd(META1_5238) gene promoter such thatprsThe transcription level is reduced to about 30% of the original transcription level. The obtained engineered strain (WT 03) had significantly higher growth capacity under low concentration methanol conditions than the wild-type strain (WT) (fig. 5).
Example 4 mutant phosphoribosyl pyrophosphate kinase in Methylobacillus torvus up-regulates the metabolic flux of phosphoketolase metabolic pathway, central metabolism is more efficient and balanced
In this example, a mutant strain and a wild strain of a mutant strain of the phosphoribosyl pyrophosphate kinase gene of a model strain of a methylotrophic bacterium belonging to the phylum alpha-Proteobacteria were cultured under 10 mM methanol carbon source conditions, and the strain was cultured at the middle-late stage (OD 600 =0.32), total RNA extraction was performed using a TransZol Up kit, followed by removal of genomic DNA, reverse transcription reaction, and cDNA was obtained. The transcription level of related genes such as serine circulation pathway, ethylmalonyl-coa pathway, phosphoketolase pathway, formate oxidation pathway, etc. was analyzed by RT-qPCR. By determining the mutation of the phosphoribosyl pyrophosphate kinase gene of M.torpedoThe Ct values of the target gene and the reference gene in the strain are calculated by using a delta Ct or 2-delta Ct method, and the relative expression amount change between the mutant strain and the wild strain of the phosphoribose pyrophosphatase gene of the methylobacterium wrenchii is compared. Mutation of phosphoribosyl pyrophosphate kinase reduces metabolic flux of nucleotide de novo synthesis and up regulates phosphoketolase pathway related genexfp、pta、ackAndacsthe transcription level (1.9-3.3 times) of the enzyme, thereby improving the metabolic flux of converting 6-phosphate-glucose and 5-phosphate-ribulose into acetyl coenzyme A. Meanwhile, serine circulation related genes in mutant strains of phosphoribosyl pyrophosphate kinase genes (such as:sga、eno、gck、ppcetc.) the transcription level is up-regulated by 1.5-5.5 times; ethylmalonyl-coa pathway related genes (e.g.:phaA、phaB、croR、ccr、ecm、pccAB、mcmABetc.) are up-regulated by 3.1 to 7.0 times; the genes related to the reverse glycolysis pathway and the formate transformation pathway are up-regulated at 1.5-4.2 times, which shows that the strain mutated by the phosphoribose pyrophosphatase gene of the methylobacterium torvus enhances the central metabolism by up-regulating the phosphoketolase pathway, so that methanol and fixed carbon dioxide are more efficiently assimilated under a low-concentration methanol carbon source, and the growth capacity is improved. Furthermore, it was found that genes involved in formate oxidation reaction in mutant strains of the phosphoribosyl pyrophosphate kinase gene of M.torpedofdhBy more than 30%, thereby reducing carbon dioxide release, allowing more carbon source assimilation into central metabolism (fig. 6).
EXAMPLE 5 overexpression of phosphoketolase metabolic pathway-related Gene improving the ability of Methylobacillus sprain to grow on an oligomethanolic carbon source
This example uses the overexpression plasmid pCM80 of M.torpedo, overexpressing the gene encoding phosphoketolase in the phosphoketolase metabolic pathwayxfpAnd the recombinant plasmid (pCM 80-xfp) Electrotransport to methylobacterium torvum. The obtained transformant (WT 11) was subjected to growth phenotype analysis under the condition of an oligomethanolic carbon source, and the result shows that the growth capacity of the transformant is obviously higher than that of the control strain (WT 10). Overexpression under methanol conditions of 5mM, 10 mM,15 mMxfpTwisting and demethylating rod of geneThe final biomass obtained from the bacterial transformant (WT 11) was OD, respectively 600 =0.234、OD 600 =0.407、OD 600 =0.588, 25.5%, 12.4%, 25% improvement over control strain (WT 10), respectively. This example provides genes encoding phosphoketolase enzymes in phosphoketolase metabolic pathwaysxfpAs a result of the overexpression, other genes of the pathway may also be overexpressed in the future to increase the ability to grow under the conditions of the oligomethanolic carbon source (FIG. 7).
Example 6 enhancement of the ability of phosphoketolase metabolic pathway-related genes to grow in genome copy number enhanced Methylobacillus torvus under an oligomethanolic carbon source
In this example, phosphoketolase gene was expressed in Methylobacillus wrenchii, a model strain of Methylobacillus alpha-proteolyticus by homologous recombinationxfp) Insertion of genes encoding cellulose synthase, a non-functional genecelAThe locus, the promoter is the strong promoter PmxF in the methylobacterium wrenchii, improves the copy number and the transcriptional expression level of the phosphoketolase gene on the genome of the methylobacterium wrenchii. The resulting engineered strain (WT 02) grew significantly higher than the wild strain (WT) at low methanol concentrations (fig. 8).
EXAMPLE 7 enhancement of the ability of mutant, engineered and engineered strains of Methylobacillus torvus to colonize the plant Arabidopsis She Jiding relative to the wild strain
This example shows that mutant strain of a-proteobacteria phylum methyl trophic bacteria (model bacteria methylobacterium wrenches) and engineered strain of a-proteobacteria phylum methyl trophic bacteria (model bacteria methylobacterium wrenches) have enhanced ability to colonize arabidopsis She Jiding. The mutant strain of M.torpedo, the engineered strain of M.torpedo, and the wild strain of M.torpedo, as described in example 2, 4, 7 were cultured under the condition of 5mM methanol carbon source to mid-exponential phase, and the cells were collected, centrifuged, washed, and treated with 10 mM MgCl 2 The solution adjusts the concentration of the bacterial cells to OD 600 0.5. Sterilizing Arabidopsis seeds by washing Arabidopsis seeds with sterile water for 10 min, washing with 75% ethanol for 2 min,followed by treatment with 5% sodium hypochlorite solution for 5 minutes and finally rinsing the seeds with sterile water 2-3 times. The sterilized seeds were spotted onto 1/2MS plates containing 1% sucrose using a sterile toothpick. Soaking seeds in 5 microliter of fungus solution, culturing in a greenhouse for 3 weeks, collecting seedlings above root, and placing in 10 mM MgCl containing 0.2% v/v silwet L-77 2 Shaking the solution for 1 hour, post-diluting and coating on a 123 mM methanol plate, and after 7 days, respectively counting CFU, wherein the number of cells of the colonized cells per gram fresh weight Arabidopsis thaliana is found to be more than 30% higher than that of the wild strain, which is described in the second embodiment (WT-prs EVO ) The modified strain of M.torpedo (WT 03) as described in example 4 and the engineered strain of M.torpedo (WT 02) as described in example 7 were all higher in the ability to colonize Arabidopsis She Jiding than the wild strain (WT) (FIG. 9).
The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.
Claims (6)
1. A mutant strain of the genus methylobacterium, characterized in that the mutant strain is based on a methylobacterium wrenchii, a phosphoribosyl pyrophosphate kinase of the methylobacterium wrenchii is mutated by a nucleotide sequence mutation technique, the 112 th nucleotide of the gene of the phosphoribosyl pyrophosphate kinase is mutated from G to A, the 38 th amino acid of the enzyme is mutated from D to N, so that the phosphoribosyl pyrophosphate kinase catalytic activity is reduced, and the amino acid sequence of the phosphoribosyl pyrophosphate kinase is shown in SEQ No. 1.
2. An engineered strain of methylobacterium, wherein the engineered strain is based on methylobacterium tenuis, reduces transcription level of phosphoribosyl pyrophosphate kinase gene by CRISPRi technology, and targets nucleotide 275 of phosphoribosyl pyrophosphate kinase gene of methylobacterium tenuis.
3. Use of one or a combination of two of the mutants of the genus methylobacterium of claim 1 or the engineered strains of the genus methylobacterium of claim 2 in plant phyllostachys growth colonisation.
4. Use of one or a combination of two of the mutants of the genus methylobacterium of claim 1 or the engineered strains of the genus methylobacterium of claim 2 for the preparation of a plant probiotic or a foliar bacterial fertilizer.
5. A method for improving colonization of a methylobacterium by plant phyllostachys, the method comprising first modifying the methylobacterium to one or a combination of two of the methylobacterium mutant strain of claim 1 or the methylobacterium engineered strain of claim 2; then, one or a combination of two of the mutant strain of the genus Methylobacillus as defined in claim 1 or the engineered strain of the genus Methylobacillus as defined in claim 2 is colonized on the leaf of a plant of a different family.
6. The method according to claim 5, wherein the method comprises culturing the mutant strain of the genus Methylobacillus according to claim 1 or the modified strain of the genus Methylobacillus according to claim 2 in a liquid medium, adjusting the concentration of the cells, immersing the plant seeds in the bacterial suspension, drip irrigation the roots of the plants, spraying the plant leaf, and applying the microbial inoculum for 3 weeks to obtain a colonization amount of the plant leaf methylobacterium of 10 per gram fresh weight of the phyllostachys plant 8 -10 9 CFU or more.
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| Acetate kinase-an enzyme of the postulated phosphoketolase pathway in Methylomicrobium alcaliphilum 20Z;Olga N.rozova等;Antonie van Leeuwenhoek;第108卷;第965-974 * |
| Metabolic regulation of "Ca. Methylacidiphilum fumariolicum" SolV cells grown under different nitrogen and oxygen limitations;Ahmad F. Khadem等;frontiers in Microbiology;第3卷;1-15页 * |
| 甲基营养菌代谢网络途径和代谢工程改造的研究进展;杨靖;生物加工过程;第15卷(第6期);第1-8页 * |
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