CN110452862B

CN110452862B - Pseudomonas fluorescens strain and application thereof

Info

Publication number: CN110452862B
Application number: CN201910659301.8A
Authority: CN
Inventors: 涂强; 张友明; 武玉侠; 陈汉娜; 徐佳莹; 边志龙; 姜彬
Original assignee: Dezhou Microp Bio Technology Co ltd; Shandong University
Current assignee: Dezhou Microp Bio Technology Co ltd; Shandong University
Priority date: 2019-07-22
Filing date: 2019-07-22
Publication date: 2022-06-07
Anticipated expiration: 2039-07-22
Also published as: CN110452862A

Abstract

The invention belongs to the technical field of microorganisms, and relates to a pseudomonas fluorescens strain and application thereof. A pseudomonas fluorescens strain, which is a pseudomonas fluorescens strain zc-C3-V and is named by classificationPseudomonas protegens zc-C3-V, which is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 6 and 21 months, wherein the preservation number is as follows: CGMCC No. 18011. The pseudomonas fluorescens strain zc-C3-V can produce 2-keto gluconic acid, and the fermented thalli can be recycled to be made into a biocontrol microbial inoculum, so that the environmental pollution is avoided, the resource is recycled, a new value is created, and the social benefit and the economic benefit are obvious.

Description

Pseudomonas fluorescens strain and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and relates to a pseudomonas fluorescens strain and application thereof.

Background

The Genome Shuffling technology is a technology for directionally screening mutants with specific phenotypic characteristic change by applying female parent fusion, and the protoplast fusion technology can cross the boundary of species, so that the target character is improved, and the Genome Shuffling technology has incomparable advantages compared with other breeding methods. The technology extends from molecular directed evolution to genome-level extension, extends genetic modification and modification from a single gene to the whole genome, and combines the target traits of strains in a wider and macroscopic range. This technique is similar to the construction of a genetic material communication mode similar to sexual reproduction in non-sexual-reproduction prokaryotes, so that strains with significantly improved phenotypes can be rapidly and efficiently bred through multiple rounds of random recombination.

Pseudomonas fluorescens is a gram-negative rod-shaped bacterium which is listed as one of registered microbial pesticides and fertilizer varieties by the Ministry of agriculture in China and is popularized and used nationwide. Has good control effect on plant diseases, particularly soil-borne diseases such as shatter rot, root rot, blight and the like, is an important beneficial microbial resource, and has very important significance on agricultural production. Related genes are introduced into pseudomonas fluorescens by using a gene recombination technology, so that a good microbial agricultural strain with a biocontrol function is formed.

2-keto-D-gluconic acid (2 KGA) is one of organic acids produced in large scale by adopting bacterial fermentation, and is mainly used for synthesizing food antioxidants D-Erythorbic Acid (EA) and D-Sodium Erythorbate (EN). Since the 90 s of the 20 th century, the demand of food antioxidants EN and EA in the international market is increasing, and the demand of an intermediate 2KGA in the production process is also increasing. The Pseudomonas fluorescens precursor has a close relationship with the biocontrol Pseudomonas protegens CHA0 in agriculture, but because a proper treatment mode is not available for a long time, the bacteria can only be treated as industrial waste, which causes resource waste and pollutes the environment.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provide a pseudomonas fluorescens strain which not only has an agricultural biocontrol effect, but also can be used for industrial production of 2KGA, so that 2 KGA-producing strain waste is utilized, the resource utilization rate is improved, and the environmental pollution is avoided.

In order to achieve the purpose, the invention adopts the technical scheme that: a pseudomonas fluorescens strain is a pseudomonas fluorescens strain zc-C3-V which is classified and namedPseudomonas protegens zc-C3-V, which is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 6 and 21 months, wherein the preservation number is as follows: CGMCC No. 18011.

Furthermore, the pseudomonas fluorescens strain zc-C3-V is a fusion of pseudomonas biocontrol C3 and pseudomonas fluorescens V.

Furthermore, the 16S rRNA sequence of the pseudomonas fluorescens strain zc-C3-V is shown as SEQ NO: 1 is shown.

The invention also provides application of the pseudomonas fluorescens strain zc-C3-V in producing 2-keto-gluconic acid, and the centrifuged thallus of the fermentation liquor is used for preparing a biocontrol microbial inoculum.

The pseudomonas fluorescens strain zc-C3-V can produce 2-keto gluconic acid, and the fermented thalli can be recycled to be made into a biocontrol microbial inoculum, so that the environmental pollution is avoided, the resource is recycled, a new value is created, and the social benefit and the economic benefit are obvious.

Drawings

FIG. 1 is a dilution of Pseudomonas fluorescens V protoplasts with water and SMM, respectively, 10⁶Coating plate contrast;

FIG. 2 is a dilution 10 of Pseudomonas biocontrol C3 protoplasts with water and SMM, respectively⁶Coating plate contrast;

FIG. 3 is a negative and positive control of fusion culture;

FIG. 4 is a photomicrograph of a fusant;

FIG. 5 shows OD curves of the cell culture process;

FIG. 6 is a graph comparing the inhibition of Bacillus subtilis by the starter and fusant;

FIG. 7 is a graph comparing the inhibition of arthrobacter by the outbreak and the fusant;

FIG. 8 is a graph showing the inhibition of yeast by the mycelia and the fusant.

Detailed Description

The Pseudomonas C3 Pseudomonas proteins CHA0 has stronger effect of inhibiting the growth and reproduction of the bacillus subtilis or the arthrobacter; pseudomonas fluorescens V is an industrial 2KGA producing strain, belongs to the genus Pseudomonas, and has the function of producing 2-ketogluconic acid.

Example one, the method and process for constructing pseudomonas fluorescens strain zc-C3-V of the present invention are as follows:

1. using biocontrol pseudomonas C3 and pseudomonas fluorescens V as starting bacteria, culturing the bacteria by using an improved KB culture medium to logarithmic phase, centrifuging to remove supernatant, and treating precipitates by using 20mg/ml lysozyme solution at 30 ℃ for 1.5h to obtain protoplasts.

2. Respectively carrying out gradient dilution on the protoplast by using SMM and pure water, and then coating a plate to calculate the protoplast formation rate, wherein the formula is as follows: protoplast formation rate = (number of SMM diluted bacteria-number of water diluted bacteria)/number of SMM diluted bacteria.

The protoplast formation rate of Pseudomonas fluorescens V is 75%, as shown in FIG. 1, the protoplast formation rate of Pseudomonas biocontrol C3 is 73%, as shown in FIG. 2, the protoplast formation rate of 8h is 88%; it can be seen that the cell wall of C3 is not easily ruptured. FIG. 1 and FIG. 2 are left views of a water dilution 10⁶Coated plates, right panel diluted 10 with osmo-stabilizing SMM⁶Coating the flat plate.

3. Equal amounts of C3 and V protoplasts were mixed and 0.02ml of nascent calcium phosphate and 0.5ml of PEG-6000 were shaken and mixed for three minutes, centrifuged at 4000rpm/min for 10min, the pellet was washed with smm solution, coated with regeneration plates containing kanamycin resistance and the plates were incubated at 50 ℃.

Pseudomonas fluorescens V has temperature resistance due to its ability to grow at 50 ℃ while Pseudomonas biocontrol C3 has a kanamycin resistance marker. Thus, when cultured at 50 ℃ on kanamycin-resistant plates, only the fusions are able to withstand both high temperatures and kanamycin resistance. As shown in FIG. 3, the left panel is a negative control, and the LBG medium is LB medium plus 15% sucrose; the right panel is a positive control, and the medium LBGK is LB plus sucrose and kanamycin.

4. The high temperature resistant and kanamycin resistant fusions were selected. A micrograph of the fusions is shown in FIG. 4.

Example 2 identification of Pseudomonas fluorescens Strain zc-C3-V

And (3) amplifying the 16S rRNA of the fusant by using a universal primer, and sending the amplified 16S rRNA to Qingdao Hippon Biotechnology Limited company for sequencing, wherein the sequence is shown as SEQ ID NO: 1, and then comparing the measured sequences with blast on line in an ncbi database, wherein the 16S rRNA is similar to Pseudomonas fluorescens sp. f2(2012) by 99 percent, thereby determining that the constructed fusant is the Pseudomonas fluorescens strain.

EXAMPLE 3 Pseudomonas fluorescens strain zc-C3-V acid production Capacity test

And (3) transferring all the obtained strains zc-C3-V to an LB plate by taking the pseudomonas fluorescens V as a reference, and performing seed culture on different monoclonals under the seed culture conditions of a seed culture medium (g/L): 20.0 parts of glucose, 10.0 parts of corn steep liquor, 2.0 parts of urea, KH2PO42.0 parts, MgSO4 & 7H2O 0.5.5 and pH 7.0 parts. Prepared according to the formula, and subpackaged into triangular bottles of 25ml/250 ml. Then placing the mixture between shaking tables at 30 ℃ and carrying out shaking culture at 200rpm for 16-20 hours. After the culture is finished, transferring into a fermentation medium. The fermentation formula is as follows: 18% of glucose, 2% of corn steep liquor, 4% of calcium carbonate and pH 6.7-7.0. Sterilizing in a sterilizing pot at 118 deg.C for 20 min. The fermentation culture conditions were 260rpm, and the culture was carried out at 30 ℃ for 72 hours. And calculating the acid-producing conversion rate according to the optical rotation value, and selecting the strain with higher optical rotation for fermentation and then analyzing the acid-producing performance of the strain. As can be seen from Table 1, the acid-producing ability of the strain zc-C3-V is improved to a different extent than that of the starting Pseudomonas fluorescens V except for C3-V5.

TABLE 1 Experimental results of acid-producing ability of Zc-C3-V strain

EXAMPLE 4 high Density fermentation culture experiment of the constructed Strain zc-C3-V of the present invention

The initial medium carbon source was 1% glycerol.

Seed culture medium: tryptone 1%, yeast extract 0.5%, sodium chloride 0.1%, pH 7. And (3) seed culture conditions: using 50ml shake flask to fill 20 ml of liquid for activating the glycerol tube strain overnight (12 h), wherein the inoculation amount is 1%, the temperature is 30 ℃, and the rpm is 200; then, a 500 ml shake flask is used for 100 ml liquid loading for seed liquid growth, the culture time is 8h, the inoculation amount is 1%, the temperature is 30 ℃, the rpm is 200, and when the OD600 reaches about 3.2, the 1L tetrad can is transferred, and the inoculation amount is 10%.

And (3) supplementary culture:

A1L quadruple tank is used, the liquid filling amount is 700 ml, the inoculation amount is 10%, the temperature is 30 ℃, the initial rotating speed is 200rpm, the speed is gradually adjusted to 400 rpm until the logarithmic growth is adjusted to 800 rpm in the early stage, the rotating speed is maintained at 1000 rpm in the middle and later stages, and the rotating speed is kept constant until the fermentation is finished. The air flux is 2.0L/min; the pH was controlled with 20% phosphoric acid and 28% concentrated ammonia, and maintained at about 7.0.

Fermentation initial medium: 1% of glycerol; 2% of corn steep liquor; dipotassium phosphate 0.15%; MgSO4 ∙ 7H2O 0.15%; pH 7.

A supplemented medium: the concentration of glucose liquid is 600 g/L.

After fermentation for 8 hours, sugar feeding is started, the feeding rate is 5.6 ml/h (the parameter is 3% according to the dial of the four-tank apparatus), and the glucose concentration is maintained at about 0.2% in the period. The pH was controlled at 7.

The glucose concentration was measured using an SBA-biosensor.

The experimental results are as follows:

the OD time profile during the culture is shown in FIG. 1, and the glucose concentration profile is shown in FIG. 5. The fermentation period is 44 h, the maximum OD of the thallus is 155.9, and the maximum dry weight of the thallus is 56.06 g/L. When the fermentation tank is put in (56 h), the glucose consumption is 147.8 g, the glucose consumption rate is 4.8 g/L/h, and the glucose yield of the thalli to the glucose is 0.379 g/g. The consumption amount of the nitrogen source (calculated as NH 3) was 3.781 g, and the yield of the bacterial cell to the nitrogen source (NH 3) was 14.83 g/g.

The supernatant obtained from the high density culture was used for acidogenesis, and the 2KGA concentration was found to be 125 g/L.

Example 5 biocontrol ability experiment of the strain zc-C3-V constructed by the invention

The inhibition effect of the spawn running and the strain zc-C3-V on bacillus subtilis, arthrobacter and fungi is respectively tested by a filter paper method.

The results of the inhibition experiment on Bacillus subtilis are shown in FIG. 6, in which C represents Pseudomonas antibiotica C3, zc represents Pseudomonas fluorescens V, and + represents the strain zc-C3-V. The bacteriostatic ability is arranged as c > zc > +,

as shown in FIG. 7, the inhibition effect on arthrobacter is shown in the figure, wherein zc represents Pseudomonas fluorescens V, C represents Pseudomonas biocontrol C3, and + represents a strain zc-C3-V, and the bacteriostatic ability is arranged in order of C > + > zc.

The inhibition effect on yeast is shown in figure 8, and the bacteriostatic ability is orderly arranged as + c > zc.

Through comparison of the experimental results, the bacterial inhibition capacity of the strain zc-C3-V is reduced compared with that of the parent pseudomonas bio-control strain C3, but the fungal inhibition capacity is more obvious compared with that of parents.

Therefore, the strain zc-C3-V of the invention can be used for preparing 2KGA, and the thallus obtained after fermentation liquor centrifugation can be used for preparing biocontrol microbial inoculum.

Sequence listing

<110> Shandong university

Texas Mike Biotechnology Ltd

<120> Pseudomonas fluorescens strain and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1436

<212> DNA

<213> Pseudomonas fluorescens (Pseudomonas)

<400> 1

gatccccgtg ggtaccgtcc tcccgaaggt tagactagct acttctggtg caacccactc 60

ccatggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg acattctgat 120

tcgcgattac tagcgattcc gacttcacgc agtcgagttg cagactgcga tccggactac 180

gatcggtttt gtgagattag ctccacctcg cggcttggca accctctgta ccgaccattg 240

tagcacgtgt gtagcccagg ccgtaagggc catgatgact tgacgtcatc cccaccttcc 300

tccggtttgt caccggcagt ctccttagag tgcccaccat gacgtgctgg taactaagga 360

caagggttgc gctcgttacg ggacttaacc caacatctca cgacacgagc tgacgacagc 420

catgcagcac ctgtgtcaga gttcccgaag gcacccatcc atctctggaa agttctctgc 480

atgtcaaggc ctggtaaggt tcttcgcgtt gcttcgaatt aaaccacatg ctccaccgct 540

tgtgcgggcc cccgtcaatt catttgagtt ttaaccttgc ggccgtactc cccaggcggt 600

caacttaatg cgttagctgc gccactaaaa tctcaaggat tccaacggct agttgacatc 660

gtttacggcg tggactacca gggtatctaa tcctgtttgc tccccacgct ttcgccacct 720

cagtggtcag tatcagtcca ggtggtcgcc ttcgccactg gtgttccttc ctatatctac 780

gcatttcacc gctacaacag gaaattccac caccctctac catactctag ctcgccagtt 840

ttggatgcag ttcccaggtt gagcccgggg ctttcacatc caacttaacg aaccacctac 900

gcgcgcttta cgcccagtaa ttccgattaa cgcttgcacc ctctgtatta ccgcggctgc 960

tggcacagag ttagccggtg cttattctgt cggtaacgtc aaaacagcaa ggtattaact 1020

tactgccctt cctcccaact taaagtgctt tacaatccga agaccttctt cacacacgcg 1080

gcatggctgg atcaggcttt cgcccattgt ccaatattcc ccactgctgc ctcccgtagg 1140

agtctggacc gtgtctcagt tccagtgtga ctgatcatcc tctcagacca gttacggatc 1200

gtcgccttgg tgagccatta cctcaccaac tagctaatcc gacctaggct catctgatag 1260

cgtgaggtcc gaagatcccc cactttctcc cgtaggacgt atgcggtatt agcgcccctt 1320

tcggaacgtt gtcccccact accaggcaga ttcctaggca ttactcaccc gtccgccgct 1380

gaatcaagga gcaagctccc gtcatccgct cgacttgcat gtgtagcctg ccgcca 1436

Claims

1. A pseudomonas fluorescens strain, characterized by: the strain is pseudomonas fluorescens strain zc-C3-V which is classified and namedPseudomonas protegens And is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 6 and 21 months, wherein the preservation number is as follows: CGMCC No. 18011; the pseudomonas fluorescens strain zc-C3-V has the characteristics of high temperature resistance of 50 ℃ and kana resistance; the strain is a fusion of pseudomonas fluorescens V and biocontrol pseudomonad C3; the 16S rRNA sequence of the strain is shown as SEQ ID NO: 1 is shown in the specification; the strain has the capability of producing 2-ketogluconate and inhibiting fungi.

2. Use of a pseudomonas fluorescens strain according to claim 1, wherein: the method is used for producing the 2-ketogluconic acid, and the thalli after the fermentation liquor centrifugation is used for preparing the biocontrol microbial inoculum.