# Patent

## Title
Bioengineering strain for production of novel microorganism-originated fungicides and uses thereof

## Abstract

The present invention relates to a bioengineering strain for production of novel microorganism-originated fungicides and uses thereof. The bioengineering strain for production of microorganism-originated fungicides of the present invention is obtained by transforming a phzH gene recombination expression plasmid into a strain producing phenazine-1-carboxylic acid, wherein the bioengineering strain produces phenazine-1-carboxamide. The present invention utilizes an existing strain producing phenazine-1-carboxylic acid to carry the phzH gene recombination expression plasmid, thereby achieving efficient expression of the phzH gene and transforming phenazine-1-carboxylic acid into phenazine-1-carboxamide. The present invention further discloses uses of the bioengineering strain, including a microorganism-originated fungicide produced by the bioengineering strain through fermentation, and preparation and application of the microorganism-originated fungicide. The antifungal activity of phenazine-1-carboxamide is not influenced by the acidity under which it is used, so that it has a stabilized antifungal activity and can be more effective in prophylaxis and treatment of crop diseases.

## Background
CROSS REFERENCE TO RELATED PATENT APPLICATION
The present application is the US national stage of PCT/CN2011/082531 filed on Nov. 21, 2011, which claims the priority of the Chinese patent application No. 201110347790.7 filed on Nov. 7, 2011, which application is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention pertains to the field of microorganism-originated pesticide production technologies, and more particularly, relates to a bioengineering strain for production of novel microorganism-originated fungicides and uses thereof.
BACKGROUND OF THE INVENTION
Loss attributable to crop diseases accounts for about 25-75% of the total production. For rice which is a major food crop in China, the annual plantation area is about 0.4 billion Mu. On the basis that the yield per Mu is 400 kg and the average reduction in output due to rice diseases is 10%, the annual economic loss will be as much as more than 30 billion RMB. As such, production of food crops is seriously threatened. So far, in addition to use of good seeds and modification of cultivation measures, plant diseases are mainly controlled by spraying chemical fungicides. Most of existing chemical fungicides are toxic and harmful to human body and animals to different degrees, and harmful components left over on the edible parts of plants result in potential threat to human health. This has received attention from the government and all levels of the society. Moreover, some chemical pesticides are hard to decompose, so that they are accumulated in the ecological system for a long period of time and pollute the environment, which is unfavorable to the sustainable developed of the social economy. Also, existing chemical pesticides are not very effective against some plant diseases. Therefore, while developing a new generation of chemical pesticides with endeavor, great efforts shall be put to study and develop high-performance biogenic pesticides that are safe, economic and highly compatible with the environment. At present, few types and amounts of biogenic pesticides are promoted and used in the production, and some types of biogenic pesticides result in resistance in plant pathogens due to long-time use, so that control efficiency is not ideal. In the case of rice sheath blight, pesticides for its control mainly depend on Jinggangmycin which is an old biogenic pesticide. However, after long-period use of nearly 40 years, some anastomosis groups of Rhizoctonia solani have become drug resistant. Moreover, Jinggangmycin is only effective against Rhizoctonia solani and has no obvious control effect against other pathogens, so it is greatly limited in respect of application scope.
A bio-pesticide growth-promoting antagonistic bacterium strain M18 has a high-efficiency, safe and broad-spectrum fungicidal effect on plant diseases. Also, it is well compatible with the environment and easily decomposed in the environment. The growth-promoting antagonistic bacterium strain M18 has been deposited at China General Microbiological Culture Collection Center, China Committee for Culture Collection of Microorganisms, a depository authority assigned by the State Intellectual Property Office of the PRC, the deposition No. being CGMCC NO.0462. Preparation and use of the bio-pesticide growth-promoting antagonistic bacterium M18 have been patented by the state with the patent no. ZL00119857.2. However, the bio-pesticide growth-promoting antagonistic bacterium strain M18 is a living bacterium agent whose mechanism mainly involves synthesis of an active component against plant diseases by strain M18 active bacteria to inhibit pathogens in crops, and the content of the synthesized active component is easily influenced by the metabolic regulation mechanism of the bacterium itself and environmental conditions. Therefore, M18 has a disadvantage of unstable control effect against plant diseases, so that it is hard for strain M18 to be applied in a large scale in agricultural production.
It has been proved that, the main active component of the growth-promoting antagonistic bacterium M18 for control of plant diseases is phenazine-1-carboxylic acid. Phenazine-1-carboxylic acid is extracted from the fermentation broth of growth-promoting antagonistic bacterium M18 to control crop diseases using the active component rather than the living bacterium, which also features high efficacy, safety, broad-spectrum and good compatibility with the environment. Also, this can overcome the deficiency that the growth-promoting antagonistic bacterium M18 is unstable in controlling diseases. However, synthesis of the active component phenazine-1-carboxylic acid through fermentation of the growth-promoting antagonistic bacterium M18 has a low titer which is only about 200 mg/L. How to increase fermentation titer and reduce cost becomes a bottleneck for development of this product. In recent years, we have made in-depth study on the regulation mechanism of synthesis of phenazine-1-carboxylic acid by the growth-promoting antagonistic bacterium M18 using molecular biological technologies. Based on this, through the means of genetic engineering, the two-component regulatory gene gacA in the genome of the growth-promoting antagonistic bacterium M18 is inactivated and mutated in a targeted manner, thereby obtaining M18G, a strain derived from M18. M18G greatly increases the yield of phenazine-1-carboxylic acid, so that the fermentation titer of phenazine-1-carboxylic acid reaches about 1500-1700 mg/L. The technical method of this study had been disclosed in Acta Microbiologica Sinica, Vol. 44, p 761-765 in 2004, and the title of the thesis is Differential Regulation of Phenazine-1-Carboxylic Acid and Pyoluteorin Production Mediated by Inactivated gacA in Pseudomonas sp. M18. In 2006, Chinese Patent (Patent No.: ZL200610023459.9) entitled Method for Preparation of Fungicides Using Growth-promoting Antagonistic Bacterium M18 provided a method for preparation of fungicides using growth-promoting antagonistic bacterium M18's derivative strains M18G and M18R, where the fungicide is prepared from the metabolites of microorganisms rather than living microorganisms, and the objective of increasing control effect is achieved by combination of the metabolites of the two derivative strains. In 2009, we further invented a method for production of phenazine-1-carboxylic acid by using bioengineering strain M18G carrying plasmid pME6032Phz, so that the fermentation titer of phenazine-1-carboxylic acid reaches 5700-6600 mg/L, which further reduces production cost and realizes large-scale application in agricultural production. This technology has been patented by the state with Patent No. ZL200910198664.2. Microorganism-originated fungicides with phenazine-1-carboxylic acid as a main component have been named Shenqinmycin by the Chinese pesticide denomination authority. Technical-grade Shenqinmycin and 1% Shenqinmycin suspension concentrate were granted with official registration nos. (Registration Nos.: PB20110314 and PB20110315) by the Ministry of Agriculture of the PRC in 2011. Shenqinmycin has been listed as one of the products promoted in the whole country during the 12th Five-Year Plan (Certificate No.: TG2011-002).
However, the control effect of phenazine-1-carboxylic acid against pathogens is closely related to the acidity (pH) under which it is used, and at pH 7.0, the antifungal activity of phenazine-1-carboxylic acid is only 20% of that at pH 5.0, so that the control effect of phenazine-1-carboxylic acid under alkaline conditions is greatly reduced. It is also found that, in the genomes of the growth-promoting antagonistic bacterium M18 and its derivative strain M18G, the phzH gene is a mutated and inactivated gene.
SUMMARY OF THE INVENTION
An objective of the present invention is, in view of the deficiencies of the prior art, to provide a bioengineering strain for production of novel microorganism-originated fungicides and application technologies thereof.
The present invention utilizes a strain producing phenazine-1-carboxylic acid to carry a recombination expression plasmid that can express phzH gene which is encoded to produce PhzH (glutamine phenazine-1-carboxylic acid amidotransferase), complement and add the copy number of the phzH gene to express in the engineering stain, thereby transforming phenazine-1-carboxylic acid into phenazine-1-carboxamide. The antifungal activity of phenazine-1-carboxamide is not influenced by the acidity under which it is used, so that it has a stabilized antifungal activity and can be more effective in prophylaxis and treatment of crop diseases.
The present invention first discloses a bioengineering strain for production of microorganism-originated fungicides, which is obtained by transforming a phzH gene recombination expression plasmid into a strain producing phenazine-1-carboxylic acid, wherein the bioengineering strain produces phenazine-1-carboxamide.
The present invention further discloses a method for constructing the bioengineering strain for production of microorganism-originated fungicides, comprising steps of:
1) amplifying a phzH gene segment;
2) inserting the amplified phzH gene segment into an expression vector to construct a phzH gene recombination expression plasmid;
3) transferring the constructed phzH gene recombination expression plasmid into a strain producing phenazine-1-carboxylic acid to construct the bioengineering strain for production of microorganism-originated fungicides.
The phzH gene recombination expression plasmid is a recombination expression plasmid cloned with a phzH gene segment. The phzH gene recombination expression plasmid can express the phzH gene in the strain producing phenazine-1-carboxylic acid where the phzH gene is encoded to produce PhzH (glutamine phenazine-1-carboxylic acid amidotransferase).
The phzH gene of the present invention comprises a coding region of the phzH gene and a noncoding region thereof.
The phzH gene segment may be a complete phzH gene or part of the phzH gene. The phzH gene segment of the present invention shall at least comprise a complete coding region of the phzH gene. Preferably, to be efficiently expressed into PhzH, the phzH gene segment comprises a complete coding region of the phzH gene and its 5′-end noncoding region. The 5′-end noncoding region comprised in the phzH gene segment may be a partial or complete 5′-end noncoding region of the phzH gene. The 5′-end noncoding region of the phzH gene comprised in the phzH gene segment shall facilitate expression of PhzH. Preferably, the 5′-end noncoding region comprised in the phzH gene segment comprises a polynucleotide segment starting from the first base upstream of the phzH gene translation initiation codon to the 683th base upstream of the codon.
Further, the PhzH is a PhzH of Pseudomonas, and the phzH gene segment is a phzH gene segment of Pseudomonas. 
Preferably, the pseudomonad is Pseudomonas aeruginosa or Pseudomonas chlororaphis. Specifically, the pseudomonad is selected from Pseudomonas aeruginosa strains PAO1, LESB58, PA14, PUPa3, or a Pseudomonas chlororaphis stain PCL1391.
Further, the PhzH is from the Pseudomonas aeruginosa strain PAO1. As illustrated in the embodiments, the amino acid sequence of PhzH is SEQ ID NO: 1.










MCGLAGWVDY TRKLDDEFPA IFAMTDTLAL 
 60



RGPDAEGIWK HRNALLGHRR LAVIDLSGGV




 



QPMSYRFPTG QEVTLVYTGE VYNHDALRER 
120



LRRAGHEFRT RSDTEVVLHA YLQWGERCCE




 



YLTGMFAFAV FDGRDGHLLL VRDRLGIKPL 
180



YYARHREGLL FGSEIKSILA HPEFAARLDA




 



VGLVDLLTLS RGTSQTPFRE VQELLPGHLL 
240



SWRPNSQAKL RRYWEVRRQE HADDLQSTVQ




 



RTRELVTRAL GAQLHADVPV CSLLSGGLDS 
300



TALTGIAQRI AKAEHGGDIN SFSVDFVGQA




 



EQFRSDDLRP DQDQPFALLA AQYIGSRHRT 
360



VLIDNAELVC ERAREEVFRA KDVPFTFGDM




 



DTSLHLMFGE IRRHSTVAIS GEGADELFGG 
420



YGWFRDPQAV AAARFPWASR VRLPAGFIDA




 



GFNRRCDLLQ YQQASYDDGL RQVEHLAGDS 
480



PEERRMREFS HLHLKRWMVL LLERKDRLSM




 



CNGLEVRVPY TDHELVEYVY NVPWSIKSRD 
540



GEEKWLLKRA CADYVPEAVL KRRKSPYPTS




 



ANLGYERFLR GSVRRLLEDA VNPVFGIVSR 
600



EFLAAELEHP EGYFNTQVSR HNLETALALE




 



GWLRLYGLSA
610






Further, the phzH gene segment is from the genome of the Pseudomonas aeruginosa strain PAO1. As illustrated in the embodiments, the base sequence of the phzH gene segment is SEQ ID NO: 2.








gtccgaggac ccgtgcagcg ggccggtgtt cggtccgtcg 
  60


acctgcgaat gcccttgagg



 


taggtcgtct ggcgggcccg gtgcagcggg cccgcttccg 
 120


gatgtatcgc tcgctcgaag



 


ttgccttctt taattctcca ttccccgcgc cgccctactt 
 180


ttcccgctcg tccatcgtcg



 


cgtcgaacgt tgccacgaaa tcagcgtcga tggacaactc 
 240


ttatattcaa tagttgtacg



 


atctgagttt gttgtagtca tttgcttagt tggctattca 
 300


tatgattgcc gtaaagcaac



 


tataagttta attggattag ccttctaagt ctttggaaag 
 360


agccgtgaac gaccctgtaa



 


tatctggttg tagagccgcg taatgatgtt tcaggatatt 
 420


tcattaattt tgtagattat



 


tgtttttcct ttgttttttt aaaaacagct accagattta 
 480


gatagatatt aattaactcg



 


gccacgtttt ttcctgttct atcattggcc ttccttgggc 
 540


gcaggcctgc cgaaactgct



 


tatcttcagg tcctcgaaaa gttcatacat cgaccgcctt 
 600


gggcgaagca ttcgtacgcc



 


ggaaatctgt ccggccgcac ggatgttttc agcatgttct 
 660


ctggatgagt ttcccgataa



 


acatcaatta gaggagtttc cctatgtgcg gtctcgcggg 
 720


ttgggtggat tacacgcgca



 


agctcgacga cgaatttccg gcgatcttcg ccatgaccga 
 780


tacgctcgcc atgcgcgggc



 


cggatgccga gggcatctgg aagcaccgca acgccctgct 
 840


gggtcaccgg cggctggcgg



 


tcatcgacct cagcggcggc gtgcagccga tgtcctatcg 
 900


ctttcccacc ggccaggagg



 


tcaccctcgt ctacaccggc gaggtgtaca accacgatgc 
 960


cctgcgcgag cggttgcgcc



 


gggccggaca tgagttccgc acccgcagcg ataccgaggt 
1020


ggtcctgcac gcctatctgc



 


aatggggcga gcgttgttgc gagtacctga ccgggatgtt 
1080


cgccttcgcc gtcttcgatg



 


gccgcgacgg ccacctgctg ctggtgcgcg accgcctggg 
1140


catcaagccg ctgtattacg



 


cgcggcaccg cgagggactg ctgttcggct cggagatcaa 
1200


gtccatcctg gcgcatccgg



 


aattcgccgc caggctcgac gcggtcggcc tggtcgacct 
1260


cctgacgctg tcccggggca



 


cttcgcagac gccgttccgc gaggtccagg aactgctgcc 
1320


cggccacctg ctgtcctggc



 


gtcccaattc ccaggcgaag ttgcgccgct attgggaggt 
1380


acgccgccag gagcatgccg



 


acgacctgca gagcaccgtg cagcgcaccc gcgaactggt 
1440


cacccgcgcc ctgggggcgc



 


aattgcacgc cgacgttccg gtgtgttcgc tgctatcggg 
1500


tgggctcgat tcgaccgccc



 


tgaccggcat cgcccagcgc atcgcgaagg cggagcacgg 
1560


cggcgacatc aattcattct



 


cggtggactt cgtcggccag gccgagtagt tccgcagcga 
1620


cgacctgcgt cccgaccagg



 


accagccgtt cgccctgctg gccgcgcagt acatcggcag 
1680


ccgtcatcgc accgtgctca



 


tcgacaatgc cgaactggtc tgcgaacgag cgcgcgaaga 
1740


ggtattccgg gccaaggacg



 


tacctttcac cttcggcgac atggatacct cgctgcacct 
1800


gatgttcggc gagatccgcc



 


ggcattccac ggtggccatc tccggtgaag gcgccgacga 
1860


gctgttcggt ggctacggct



 


ggttccgcga tccgcaggcg gtggctgcgg cgcgcttccc 
1920


ctgggcctcc agggtgcgcc



 


tgccagccgg cttcatcgac gccggtttca accgccgctg 
1980


cgatctcctc cagtaccagc



 


aggccagcta cgacgatggg ctgcgccagg tcgaacacct 
2040


ggccggcgac agcccggagg



 


agcggcggat gcgcgagttc agccacctgc atctgaagcg 
2100


ctggatggtg ctgctgctcg



 


aacgcaagga tcgcctgagc atgtgcaacg gcctggaggt 
2160


gcgggtgccc tacaccgacc



 


atgagctggt ggagtacgtc tacaacgtgc cctggtcgat 
2220


caagagccgg gacggcgagg



 


agaagtggct gctcaagcgg gcctgcgccg actatgtccc 
2280


ggaagccgtg ctcaagcgcc



 


gcaagagccc ttatccgact tctgccaacc tcggctacga 
2340


gcgtttcctg cgcgggagcg



 


tgcggcgtct gctggaggac gcggcgaacc cggtgttcgg 
2400


catcgtttcg cgagagttcc



 


tggccgccga actggagcat ccggaggggt acttcaacac 
2460


ccaggtgagc cgccacaacc



 


tggagaccgc gctggcgctg gaaggctggc tcaggttgta 
2516


cgggctctcc gcctga







The underlined and bold codons in the base sequence are the initiation codon and termination codon of the phzH gene respectively, and the bases before the initiation codon constitute the 5′-end noncoding region of the phzH gene.
Further, the expression vector for constructing the phzH gene recombination expression plasmid is an Escherichia coli/Pseudomonas shuttle expression plasmid.
In order that the phzH gene recombination expression plasmid can correctly express the phzH gene, it shall ensure that the phzH gene reading frame at the multiple clone site of the Escherichia coli/Pseudomonas shuttle expression plasmid is correct.
Preferably, to promote efficient expression of PhzH, the Escherichia coli/Pseudomonas shuttle expression plasmid comprises a strong promoter, the phzH gene segment is cloned behind the strong promoter, and the expression of the phzH gene segment is controlled by the strong promoter. The strong promoter may be a phage promoter T3 prom or T7 prom.
Insertion of a target gene in a specific site of the plasmid and ensuring the correct reading frame of the target gene are technologies well known by those skilled in the art. The Escherichia coli/Pseudomonas shuttle expression plasmid includes plasmids of pBBR1MCS series and derivative expression plasmids thereof. As illustrated in the embodiments, the Escherichia coli/Pseudomonas shuttle expression plasmid is pBBR1MCS-5. With a proper design of primers, the amplified phzH gene segment is under the control of the phage promoter T3 prom of pBBR1MCS-5 while ensuring the correct reading frame, thereby obtaining the gene recombination expression plasmid pBBRphzH.
The strain producing phenazine-1-carboxylic acid refers to a wild strain that produces phenazine-1-carboxylic acid through fermentation, as well as its derivative engineering strains. Further, the strain producing phenazine-1-carboxylic acid belongs to Pseudomonas, such as M18 and M18G.
The M18, with Deposition No. CGMCC NO.0462, is a bio-pesticide growth-promoting antagonistic bacterium, and phenazine-1-carboxylic acid, an active component for controlling plant diseases, can ben synthesized by living M18. M18 belongs to the prior art.
The M18G strain is a derivative strain of M18 (CGMCC NO.0462) and belongs to the prior art. The preparation method of M18G is known, for example, from Differential Regulation of Phenazine-1-Carboxylic Acid and Pyoluteorin Production Mediated by Inactivated gacA in Pseudomonas sp. M18, Acta Microbiologica Sinica, Vol. 44, p 761-765 in 2004. As compared with M18, the yield of phenazine-1-carboxylic acid in the case of M18G is greatly increased. Therefore, M18G is the preferred engineering strain producing phenazine-1-carboxylic acid in the present invention.
The wild strain producing phenazine-1-carboxylic acid and its derivative engineering strains can produce phenazine-1-carboxylic acid, the phzH gene recombination expression plasmid can express the phzH gene in the engineering strain producing phenazine-1-carboxylic acid, and the glutamine phenazine-1-carboxylic acid amidotransferase which is an encoding product of the phzH gene can amidate phenazine-1-carboxylic acid to synthesize phenazine-1-carboxamide (the molecular structural formula of phenazine-1-carboxamide is shown in FIG. 1). As such, the metabolites of the bioengineering strain of the present invention comprise phenazine-1-carboxamide.
The phzH gene recombination expression plasmid is introduced into the engineering strain producing phenazine-1-carboxylic acid by a conventional transformation or transduction method.
The bioengineering strain for production of microorganism-originated fungicides of the present invention can be used in production of the microorganism-originated fungicide of the present invention through fermentation.
The present invention further provides a microorganism-originated fungicide which is a fermentation broth of the bioengineering strain of the present invention.
The main fungicidal active component in the fermentation broth is phenazine-1-carboxamide. Also, the fermentation broth comprises a trace amount of pyoluteorin as a fungicidal active component.
Further, in the fermentation broth, the content of phenazine-1-carboxamide is 2500-2800 mg/L.
The present invention further discloses a preparation method of the microorganism-originated fungicide, where the microorganism-originated fungicide is obtained by fermenting and culturing the bioengineering strain of the present invention under conditions suitable for expression of phenazine-1-carboxylic acid and PhzH.
Further, the preparation method of the microorganism-originated fungicide comprises steps of:
1. activation of bioengineering strain;
2. inoculum enlargement: shake-flask culturing in a glycerol culture medium first and then transferring to a bacteriocin-producing culture medium for enlarged fermentation culturing to obtain the fermentation broth.
The activation of bioengineering strain may be conducted using a solid glycerol culture medium.
The glycerol culture medium comprises the following components in their respective weight percentages: 1.8-2.2% of peptone, 1.3-1.7% of glycerol, 0.05-0.1% of magnesium sulfate, 0.01-0.05% of potassium dihydrogen phosphate, the balance being water, pH 6.8-7.2.
The solid glycerol culture medium comprises the following components in their respective weight percentages: 1.8-2.2% of peptone, 1.3-1.7% of glycerol, 0.05-0.1% of magnesium sulfate, 0.01-0.05% of potassium dihydrogen phosphate, 1.2-1.5% of agar, the balance being water, pH 6.8-7.2.
The bacteriocin-producing culture medium comprises the following components in their respective weight percentages: 2.2-3.0% of peptone, 2.0-2.5% of glucose, 0.5-0.7% of potassium nitrate, the balance being water, pH 6.5-7.0.
Preferably, the engineering strain is activated as follows: the engineering strain is inoculated onto a plate with the glycerol culture medium where the engineering strain is activated and grows at 26-30° C. for 20-24 h; then, the strain mass is again streaked onto a plate with the glycerol culture medium, and activated at 26-30° C. for 10-12 h.
Preferably, in the inoculum enlargement, the shake-flask culturing in the glycerol culture medium is conducted as follows: the activated stain is inoculated into the glycerol culture medium where the strain is shake-cultured at 26-30° C. for 9-11 h. In shake-culturing, the rotation speed of the shaking table may be 160-180 rpm.
Preferably, in the inoculum enlargement, the enlarged fermentation culturing in the bacteriocin-producing culture medium is conducted as follows: the shake-cultured stain in the glycerol culture medium is transferred into the bacteriocin-producing culture medium where the strain is fermentation-cultured at 26-30° C. for 60-72 h. In fermentation culturing, the rotation speed of the shaking table may be 160-180 rpm.
The microorganism-originated fungicide of the present invention may be used in prophylaxis and treatment of plant diseases or in preparation of pesticides for prophylaxis and treatment of plant diseases.
Further, the microorganism-originated fungicide of the present invention is applied to crops by spraying or root irrigation for prophylaxis and treatment of rice sheath blight, rice bacterial leaf blight, rice false smut, wheat scab, cucumber blight disease, watermelon blight disease, melon gummy stem blight, cotton blight disease, anthrax, drooping disease, and plants diseases caused by Pythium or Phytophthora. 
The present invention further discloses a pesticide for prophylaxis and treatment of plant diseases, which comprises a fungicidally effective amount of the microorganism-originated fungicide of the present invention or its fungicidal active components.
The fermentation broth of the bioengineering strain for production of microorganism-originated fungicides of the present invention may be directly used as a pesticide for prophylaxis and treatment of plant diseases. Also, the fermentation broth may also be dried into powder by a conventional method to be used as a fungicidal active component raw material to prepare pesticides for prophylaxis and treatment of plant diseases together with other conventional adjuvants. Alternatively, the major fungicidal active component phenazine-1-carboxamide in the fermentation broth may be isolated first and then used as a fungicidal active component raw material to prepare pesticides for prophylaxis and treatment of plant diseases.
The method of the present invention involving producing phenazine-1-carboxamide by utilizing an genetic engineering strain producing phenazine-1-carboxylic acid to carry a recombination expression vector has the following advantages:
1. The antifungal activity is stable and not influenced by acidity. The phenazine-1-carboxamide produced by the present invention not only has a high antifungal ability, but also is not influenced by the acidity of the environment at pH ranging from 4.0-8.0, so that it can provide a stable effect of prophylaxis and treatment whether it is used in crops planted in an acidic environment or in an alkaline environment.
2. Transformation efficiency is high. In the present invention, phenazine-1-carboxylic acid in the host is transformed into phenazine-1-carboxamide by constructing a gene recombination plasmid carrying the complete coding region of the phzH gene and its 5′-end noncoding region. In a preferred embodiment, the phzH gene and its 5′-end noncoding region are highly expressed under the control of the phage strong promoter T3 prom and meanwhile the recombination plasmid has multiple copies in the host M18G so that the phzH gene can be highly expressed and amidated enzyme can be synthesized in the host M18G and all phenazine-1-carboxylic acid is transformed into phenazine-1-carboxamide. As a result, the transformation efficiency may be as high as 100%.
3. Production cost is low and economic benefit is high. For production of phenazine-1-carboxamide by the present invention, apart from the one-off early cost consumed by preparation and construction of the engineering strain, once the industrialized production is started, additional incorporation of other raw materials and consumption of additional energy are not required other than consumption of culture medium. As such, with expansion and extension of production scale, the average production cost for unit yield is reduced significantly, which facilitates large-scale promotion and application in agricultural production.
4. Clean production and environment protection are achieved. Production of phenazine-1-carboxamide by a biotechnology can transform all phenazine-1-carboxylic acid into phenazine-1-carboxamide. In production, apart from consumption of culture medium, other special raw materials are not used and any other intermediates are not produced. As such, no new pollutants are produced and released into the environment, so that clear production is achieved and ecological environment is protected.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a molecular structural formula of phenazine-1-carboxamide in the present invention.
FIG. 2 is a schematic diagram for construction of the plasmid pBBRphzH in the present invention.



The present invention relates to a method for production of phenazine-1-carboxamide by utilizing an engineering stain M18G to carry plasmid pBBRphzH, where the segment of the phzH gene and its 5′-end noncoding region is amplified from the PAO1 genome of Pseudomonas aeruginosa, and this segment is then inserted into the expression plasmid pBBR1MCS-5 under the control of the phage promoter T3 prom so as to be constructed into pBBRphzH; the recombination plasmid is then transferred into the derivate strain M18G of the growth-promoting antagonistic bacterium M18 to construct a genetic engineering strain M18G/pBBRphzH which realizes the high and stable expression of glutamine phenazine-1-carboxylic acid amidotransferase (PhzH). Finally, the genetic engineering strain M18G/pBBRphzH is cultured in the culture medium to efficiently and stably produce phenazine-1-carboxamide. For the phenazine-1-carboxamide prepared by using the high-yield genetic engineering strain of the present invention, the effect of prophylaxis and treatment of disease is not influenced by acidity and its production cost is further reduced. Also, it is effective in prophylaxis and treatment of plants diseases whether it is used under acidic or alkaline conditions.
FIG. 3 shows the results of comparing the antifungal activities of phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) of the same concentration against rhizoctonia solani in potato glucose culture media under different acidities. In the experiment, the weight percentages of the components in the potato glucose culture medium are as follows: 20% of potato, 2% of glucose, 1.2% of agar powder, the balance being water.
DETAILED DESCRIPTION OF THE INVENTION
A preferred embodiment of the present invention utilizes the derivative strain M18G of the growth-promoting antagonistic strain M18 to carry the recombination plasmid pBBRphzH to construct the genetic engineering strain M18G/pBBRphzH, where the copy number of the segment of the complete coding region of the phzH gene and its 5′-end noncoding region is complemented and added to transform phenazine-1-carboxylic acid into phenazine-1-carboxamide, thereby producing phenazine-1-carboxamide.
According to the preferred embodiment of the present invention, the segment of the complete coding region of the phzH gene and its 5′-end noncoding region is amplified from the PAO1 of Pseudomonas aeruginosa strains, and this segment is then inserted into the expression plasmid pBBR1MCS-5 so as to construct a recombination plasmid pBBRphzH while the segment is under the control of the phage promoter T3 prom; the recombination plasmid pBBRphzH is then transferred into the derivate strain M18G of the growth-promoting antagonistic bacterium M18 to construct a genetic engineering strain M18G/pBBRphzH which realizes the high and stable expression of the phzH gene. Finally, the genetic engineering strain M18G/pBBRphzH is cultured in the culture medium to efficiently and stably produce phenazine-1-carboxamide instead of phenazine-1-carboxylic acid. The yield of phenazine-1-carboxamide reaches a level of 2500-2800 mg. Under pH equal to or greater than 7.0, the activity of phenazine-1-carboxamide against rice sheath blight is increased by more than 5 times relative to phenazine-1-carboxylic acid. As reckoned by this antifungal activity, the antifungal activity of phenazine-1-carboxamide produced per unit volume of the fermentation broth considerably exceeds the antifungal activity of phenazine-1-carboxylic acid.
A specific plan for constructing the genetic engineering strain M18G/pBBRphzH and utilizing this strain to produce phenazine-1-carboxamide is as follows:
1. Amplification of the Segment of the phzH Gene and its 5′-end Noncoding Region
A pair of primers are designed, and the nucleotide sequences of the primers are as follows:









Forward:



(SEQ ID NO: 3)



5′-CGCGCTCGAGGTCCGAGGACCCGTGCAGC-3′



 



Reverse:



(SEQ ID NO: 4)



5′-CGCGAAGCTTTCAGGCGGAGAGCCCGTAC-3′






What are underlined in the sequences are cutting sites of restriction enzymes Xho I and Hind III; then with the PAO1 genomic DNA of Pseudomonas aeruginosa strains as a template, the segment of the phzH gene and its 5′-end noncoding region is amplified by using DNA polymerase LA Taq and the designed primers, and the amplification product is detected by agarose electrophoresis. The segment of the phzH gene and its 5′-end noncoding region of 2.5 kb long is recovered.
2. Construction of Recombinant Plasmid pBBRphzH
The recovered gene amplification segment phzH and its 5′-end noncoding region are digested by restriction enzymes Xho I and Hind III, and by the action of ligase, inserted into the corresponding cutting sites in the Escherichia coli/Pseudomonas shuttle expression plasmid pBBR1MCS-5, so that expression of the complete coding region of the phzH gene and its 5′-end noncoding region is under the control of the phage promoter T3 prom, thereby forming the recombination plasmid pBBRphzH which is then transformed into Escherichia coli; on a gentamicin resistant plate, Escherichia coli transformants transformed with pBBRphzH are screened out; the constructed gene recombination plasmid pBBRphzH is extracted from Escherichia coli. 
3. Construction of Genetic Engineering Strain M18G/pBBRphzH
The competent cells of the derivative strain M18G of the growth-promoting antagonistic strain M18 are prepared, and the aforesaid gene recombination plasmid pBBRphzH is transformed into the competent cells of M18G which are then cultured at 28-37° C. for 1-2 d. The genetic engineering strain M18G/pBBRphzH is screened out from the culture.
4. Culturing of Genetic Engineering Strain M18G/pBBRphzH
The genetic engineering strain M18G/pBBRphzH is inoculated onto a plate with a glycerol culture medium where M18G/pBBRphzH is activated and grows at 26-30° C. for 20-24 h; then, the fungal mass is again streaked onto a plate with a glycerol culture medium and activated at 26-30° C. for 10-12 h. Then, the activated M18G/pBBRphzH fungal mass is transferred into a 250 ml triangular flask containing 25 ml of a glycerol culture medium, and shake-cultured on a shake table at 26-30° C. for 9-11 h, the rotation speed of the shaking table being 160-180 rpm; finally, the strain is transferred into a 500 ml triangular flask containing 65 ml of a bacteriocin-producing culture medium where the strain is enlarged fermentation-cultured for 60-72 h, the temperature and rotation speed being unchanged, thereby obtaining phenazine-1-carboxamide with a yield of 2500-2800 mg/L.
The present invention is to be further described below in conjunction with examples. It shall be understood that, these examples are only for the purpose of explaining the present invention rather than limiting the scope of the present invention. In the examples below, the experiment methods for which the specific conditions are not described and the reagents for which the formulas are not provided are all conducted or prepared according to conventional conditions such as conditions as described in Molecular Cloning: A Laboratory Manual or conditions as recommended by the manufacturer. The examples below are not limitations to the present invention.
Example 1
1. Amplification of Segment of the phzH Gene and its 5′-end Noncoding Region
A pair of primers were designed for amplification of the segment of the phzH gene and its 5′-end noncoding region, and the nucleotide sequences of the primers were as follows:









Forward:



5′-CGCGCTCGAGGTCCGAGGACCCGTGCAGC-3′



 



Reverse:



5′-CGCGAAGCTTTCAGGCGGAGAGCCCGTAC-3′






The underlined nucleotides in the sequences were cutting sites of restriction enzymes Xho I and Hind III. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
Then, with the PAO1 genomic DNA of Pseudomonas aeruginosa strains as a template, the segment of the phzH gene and its 5′-end noncoding region was amplified by using the DNA polymerase LA Taq and the aforesaid designed primers, and the amplification product was detected by 0.7% agarose electrophoresis. The segment of the phzH gene and its 5′-end noncoding region of 2.5 kb long was recovered. The gene segment was verified correct by nucleotide sequencing. Specifically, the PAO1 genomic DNA of Pseudomonas aeruginosa was prepared by using an AxyPrep bacterial genomic DNA kit, and the gene segment was recovered using an AxyPrep DNA gel recovery kit. Both of the kits were provided by Axygen Biotechnology (Hangzhou) Co., Ltd with product catalog nos. of AP-MN-BT-GDNA-4 and AP-GX-50 respectively. The gene amplification and agarose electrophoresis were conducted according to the methods as described in p 611-618, Chapter 8 and p 387-400, Chapter 5 of Molecular Cloning: A Laboratory Manual (3rd edition) published by Science Press in 2002. Specifically, the DNA polymerase LA Taq and gene amplification kit was purchased from Shanghai Agency of TAKARA with a product catalog no. of DRR002AG, and the agarose was purchased from Shanghai Agency of GENE TECH. The nucleotide sequencing of the gene segment (phzH and its 5′-end noncoding region) was assigned to and completed by Invitrogen (Shanghai) Biotech Co., Ltd., and the sequencing results confirmed that the gene segment comprised SEQ ID NO: 2, and the amino acid sequence of the protein encoded by SEQ ID NO: 2 was SEQ ID NO: 1.
2. Construction of Recombinant Plasmid pBBRphzH
The recovered gene amplification segment (phzH and its 5′-end noncoding region) was digested by restriction enzymes Xho I and Hind III, and by the action of a ligase, inserted into the corresponding cutting sites in the Escherichia coli/Pseudomonas shuttle expression plasmid pBBR1MCS-5; expression of the segment of the phzH gene and its 5′-end noncoding region was under the control of phage promoter T3 prom, thereby forming a recombination plasmid pBBRphzH which was then transformed into Escherichia coli. On a gentamicin resistant plate, Escherichia coli transformants transformed with pBBRphzH were screened out. Finally, the constructed gene recombination plasmid pBBRphzH was extracted from the Escherichia coli transformants and verified.
The constructed gene recombination plasmid pBBRphzH is as shown in FIG. 2, where the segment of the complete coding region comprising the phzH gene and its 5′-end noncoding region is digested by restriction enzymes Xho I and Hind III, and by the action of a ligase, inserted into the corresponding cutting sites in the Escherichia coli/Pseudomonas shuttle expression plasmid pBBR1MCS-5 to be under the control of the phage promoter T3 prom, thereby forming the recombination plasmid pBBRphzH. In FIG. 2, 4766 bp represents the length of the plasmid pBBR1MCS-5, which includes 4766 base pairs. In the figure, phzH represents glutamine phenazine-1-carboxylic acid amidotransferase encoding gene; // is a symbol indicating shortened gene segment; Xho I and Hind III are restriction enzyme cutting sites; and numerals in the figure indicate the lengths of the encoding sequence of the phzH gene and the nucleotide sequence of its 5′-end noncoding region ligated with plasmid pBBR1MCS-5, which are 1833 bp and 638 bp respectively; Gm is the selective marker gene resistant to gentamicin in the plasmid pBBR1MCS-5; T3 prom and T7 prom are two phage promoters; what is marked by mob is the gene required by plasmid transfer; what is marked by rep is the sequence required by plasmid replication; the gene indicated by lacZ encodes the α-peptide chain encoded by the lacZ′ gene of Escherichia coli. 
The aforesaid targeted insertion of gene segment into plasmid, preparation and transformation of competent Escherichia coli, and extraction and verification of recombination plasmid were carried out according to the methods as described in p 68-71 and p 96-99 Chapter 1, and p 663-666, Chapter 8 of Molecular Cloning: A Laboratory Manual (3rd edition) edited by J. Sambrook and D. W. Russell and published by Science Press in 2002. Specifically, the pBBR1MCS-5 plasmid was provided by the School of Life Sciences and Biotechnology, Shanghai Jiao Tong University. The restriction enzyme and ligase were purchased from Shenzhen Zhongjing Biotechnology Co., Ltd. The recombination plasmid in Escherichia coli was extracted using a type B plasmid extraction mini kit which was provided by Beijing Biodev-tech Scientific & Technical Co., Ltd with a product catalog no. of MK014-2. The restriction enzyme, DNA polymerase LA Taq and gene amplification kits used in verification of recombination plasmid were all purchased from Shanghai Agency of TAKARA with a product catalog no. of DRR002AG. The agarose was purchased from Shanghai Agency of GENE TECH.
3. Construction of Genetic Engineering Strain M18G/pBBRphzH
The competent cells of the derivative strain M18G of the growth-promoting antagonistic strain M18 were prepared, and the aforesaid gene recombination plasmid pBBRphzH was transformed into the competent cells of M18G which were then cultured at 28° C. for 2 d. The genetic engineering strain M18G/pBBRphzH was screened out from the culture.
The preparation of the competent cells of the derivative strain M18G of the growth-promoting antagonistic strain M18, transformation of recombination plasmid pBBRphzH into the competent cells of M18G, and screening of genetic engineering strain M18G/pBBRphzH for efficient production of phenazine-1-carboxamide were carried out according to the methods as described in p 96-99, Chapter 1 of Molecular Cloning: A Laboratory Manual (3rd edition) edited by J. Sambrook and D. W. Russell and published by Science Press in 2002.
4. Culturing of Genetic Engineering Strain M18G/pBBRphzH
The genetic engineering strain M18G/pBBRphzH was inoculated onto a plate with a glycerol culture medium where M18G/pBBRphzH was activated and grew at 26° C. for 24 h; then, the strain mass was again streaked onto a plate with a glycerol culture medium and activated at 26° C. for 10 h. Then, the activated M18G/pBBRphzH fungal mass was transferred into a 250 ml triangular flask containing 25 ml of a glycerol culture medium, and shake-cultured on a shake table at 26° C. for 9 h, the rotation speed of the shaking table being 160 rpm. Finally, the strain was transferred into a 500 ml special triangular flask containing 65 ml of a bacteriocin-producing culture medium where the strain was enlarged fermentation-cultured for 60 h, the temperature and rotation speed being unchanged, thereby obtaining phenazine-1-carboxamide with a yield of 2500 mg/L in the fermentation broth. The content of phenazine-1-carboxylic acid was not detected out, indicating that all phenazine-1-carboxylic acid had transformed into phenazine-1-carboxamide. As shown by the detection results, the rate of transformation of phenazine-1-carboxylic acid into phenazine-1-carboxamide was 100%.
Specifically, the glycerol culture medium comprised the following components in their respective weight percentages: 1.8% of peptone, 1.3% of glycerol, 0.07% of magnesium sulfate, 0.03% of potassium dihydrogen phosphate, the balance being water, pH 7.0. The glycerol culture medium (solid) further comprised 1.5% of agar. The bacteriocin-producing culture medium comprised the following components in their respective weight percentages: 2.2% of peptone, 2.0% of glucose, 0.5% of potassium nitrate, the balance being water, pH6.8.
With this formula, the 65 ml of M18G/pBBRphzH fermentation broth provided 162.5 mg of phenazine-1-carboxamide, the content of phenazine-1-carboxylic acid being zero. This fermentation broth was diluted proportionally to prepare a potato glucose culture medium (pH 7.0) containing 16 mg/ml phenazine-1-carboxamide. Also, an M18G/pME6032Phz fermentation broth (recorded in ZL200910198664.2) was diluted proportionally to prepare a potato glucose culture medium (pH 7.0) containing 16 mg/ml phenazine-1-carboxylic acid. With a potato glucose culture medium (pH 7.0) without any fermentation broth as a control, the growth rates of Rhizoctonia solani in the potato glucose culture media (pH 7.0) were measured respectively and the antifungal activities of phenazine-1-carboxamide and phenazine-1-carboxylic acid were calculated according to the method as described in Example 6. For bioactivity at the same pH of 7.0, the antifungal activity of phenazine-1-carboxamide obtained per liter of the M18G/pBBRphzH fermentation broth was equivalent to that of 12500 mg of phenazine-1-carboxylic acid, the antifungal activity being increased about 1.9 times.
Example 2
1. The complete coding region of the phzH gene and its 5′-end noncoding region were amplified by using the same method as in Example 1. The product was detected by 1.0% agarose electrophoresis and recovered to obtain the segment of the phzH gene and its 5′-end noncoding region of about 2.5 kb long.
2. The recovered segment of the phzH gene and its 5′-end noncoding region was digested by restriction enzymes Xho I and Hind III, and as ligated by a ligase, inserted into the Escherichia coli/Pseudomonas shuttle expression plasmid pBBR1MCS-5; expression of the segment of the phzH gene and its 5′-end noncoding region was under the control of the phage promoter T3 prom, thereby forming the recombination plasmid pBBRphzH which was then transformed into Escherichia coli. On a gentamicin resistant plate, Escherichia coli transformants transformed with pBBRphzH were screened out. Finally, the recombination plasmid was extracted from the Escherichia coli transformants and verified.
3. The competent cells of the derivative strain M18G of the growth-promoting antagonistic strain M18 were prepared, and the aforesaid recombination plasmid pBBRphzH was transformed into the competent cells of M18G which were then cultured at 30° C. for 1 d.
The preparation of the competent cells of the derivative strain M18G of the growth-promoting antagonistic strain M18, transformation of the recombination plasmid pBBRphzH into the competent cells of M18G, and screening of the genetic engineering strain M18G/pBBRphzH for efficient production of phenazine-1-carboxamide were all carried out according to the same methods as in Example 1.
4. The genetic engineering strain M18G/pBBRphzH was inoculated onto a plate with a glycerol culture medium where M18G/pBBRphzH was activated and grew at 28° C. for 22 h; then, the strain mass was again streaked onto a plate with a glycerol culture medium and activated at 28° C. for 11 h. Then, the activated M18G/pBBRphzH fungal mass was transferred into a 250 ml triangular flask containing 25 ml of a glycerol culture medium, and shake-cultured on a shake table at 28° C. for 10 h, the rotation speed of the shaking table being 170 rpm. Finally, the strain was transferred into a 500 ml triangular flask containing 65 ml of a bacteriocin-producing culture medium where the strain was enlarged fermentation-cultured for 66 h, the temperature and rotation speed being unchanged, thereby obtaining phenazine-1-carboxamide with a yield of 2700 mg/L in the fermentation broth. The content of phenazine-1-carboxylic acid was not detected out, indicating that all phenazine-1-carboxylic acid had transformed into phenazine-1-carboxamide. As shown by the detection results, the rate of transformation of phenazine-1-carboxylic acid into phenazine-1-carboxamide was 100%.
Specifically, the glycerol culture medium comprised the following components in their respective weight percentages: 2.2% of peptone, 1.7% of glycerol, 0.05% of magnesium sulfate, 0.01% of potassium dihydrogen phosphate, the balance being water, pH 7.2. The glycerol culture medium (solid) further comprised 1.5% of agar. The bacteriocin-producing culture medium comprised the following components in their respective weight percentages: 2.2% of peptone, 2.5% of glucose, 0.5% of potassium nitrate, the balance being water, pH7.2.
With this formula, the 65 ml of M18G/pBBRphzH fermentation broth provided 175.5 mg of phenazine-1-carboxamide, the content of phenazine-1-carboxylic acid being zero. This fermentation broth was diluted proportionally to prepare a potato glucose culture medium (pH 7.0) containing 16 mg/ml phenazine-1-carboxamide. Also, an M18G/pME6032Phz fermentation broth (recorded in ZL200910198664.2) was diluted proportionally to prepare a potato glucose culture medium (pH 7.0) containing 16 mg/ml phenazine-1-carboxylic acid. With a potato glucose culture medium (pH 7.0) without any fermentation broth as a control, the growth rates of rhizoctonia solani in the potato glucose culture media (pH 7.0) were measured respectively and the antifungal activities of phenazine-1-carboxamide and phenazine-1-carboxylic acid were calculated according to the method as described in Example 6. For bioactivity at the same pH of 7.0, the antifungal activity of phenazine-1-carboxamide obtained per liter of the M18G/pBBRphzH fermentation broth was equivalent to that of 13500 mg of phenazine-1-carboxylic acid, the antifungal activity being increased about 2.1 times.
Example 3
1. The complete coding region of the phzH gene and its 5′-end noncoding region were amplified by using the same method as in Example 1. The product was detected by 1.0% agarose electrophoresis and recovered to obtain the segment of the phzH gene and its 5′-end noncoding region of about 2.5 kb long.
2. The recovered segment of the phzH gene and its 5′-end noncoding region was digested by restriction enzymes Xho I and Hind III, and by the action of a ligase, inserted into the corresponding restriction enzyme cutting site of Escherichia coli/Pseudomonas shuttle expression plasmid pBBR1MCS-5 to be under the control of the phage promoter T3 prom, thereby forming the recombination plasmid pBBRphzH which was then transformed into Escherichia coli. On a gentamicin resistant plate, Escherichia coli transformants transformed with pBBRphzH were screened out. Finally, the recombination plasmid was extracted from the Escherichia coli and verified.
3. The competent cells of the derivative strain M18G of the growth-promoting antagonistic strain M18 were prepared, and the aforesaid recombination plasmid pBBRphzH was transformed into the competent cells of M18G which were then cultured at 32° C. for 2 d, and then from which the genetic engineering strain M18G/pBBRphzH for efficient production of phenazine-1-carboxamide was screen out.
The preparation of the competent cells of the derivative strain M18G of the growth-promoting antagonistic strain M18, transformation of the recombination plasmid pBBRphzH into the competent cells of M18G, and screening of the genetic engineering strain M18G/pBBRphzH for efficient production of phenazine-1-carboxamide were all carried out according to the same methods as in Example 1.
4. The genetic engineering strain M18G/pBBRphzH constructed by the gene engineering technology was inoculated onto a plate with a glycerol culture medium where M18G/pBBRphzH was activated and grew at 30° C. for 20 h; then, the strain mass was again streaked onto a plate with a glycerol culture medium and activated at 30° C. for 12 h. Then, the activated M18G/pBBRphzH strain was transferred into a 250 ml triangular flask containing 25 ml of a glycerol culture medium, and shake-cultured on a shake table at 30° C. for 11 h, the rotation speed of the shaking table being 180 rpm. Finally, the strain was transferred into a 500 ml triangular flask containing 65 ml of a bacteriocin-producing culture medium where the strain was enlarged fermentation-cultured for 72 h, the temperature and rotation speed being unchanged, thereby obtaining phenazine-1-carboxamide with a yield of 2800 mg/L in the fermentation broth. The content of phenazine-1-carboxylic acid was not detected out, indicating that all phenazine-1-carboxylic acid had transformed into phenazine-1-carboxamide. As shown by the detection results, the rate of transformation of phenazine-1-carboxylic acid into phenazine-1-carboxamide was 100%.
Specifically, the glycerol culture medium comprised the following components in their respective weight percentages: 2.0% of peptone, 1.5% of glycerol, 0.1% of magnesium sulfate, 0.05% of potassium dihydrogen phosphate, the balance being water, pH 6.8. The glycerol culture medium (solid) further comprised 1.5% of agar. The bacteriocin-producing culture medium comprised the following components in their respective weight percentages: 3.0% of peptone, 2.5% of glucose, 0.8% of potassium nitrate, the balance being water, pH7.0.
With this formula, the 65 ml of M18G/pBBRphzH fermentation broth provided 182 mg of phenazine-1-carboxamide, the content of phenazine-1-carboxylic acid being zero. This fermentation broth was diluted proportionally to prepare a potato glucose culture medium (pH 7.0) containing 16 mg/ml phenazine-1-carboxamide. Also, an M18G/pME6032Phz fermentation broth (recorded in ZL200910198664.2) was diluted proportionally to prepare a potato glucose culture medium (pH 7.0) containing 16 mg/ml phenazine-1-carboxylic acid. With a potato glucose culture medium (pH 7.0) without any fermentation broth as a control, the growth rates of rhizoctonia solani were measured respectively and the antifungal activities of phenazine-1-carboxamide and phenazine-1-carboxylic acid were calculated according to the method as described in Example 6. For bioactivity at the same pH of 7.0, the antifungal activity of phenazine-1-carboxamide obtained per liter of the M18G/pBBRphzH fermentation broth was equivalent to that of 14000 mg of phenazine-1-carboxylic acid, the antifungal activity being increased about 2.2 times.
Example 4
1. The complete coding region of the phzH gene and its 5′-end noncoding region were amplified by using the same method as in Example 1. The product was detected by 1.0% agarose electrophoresis and recovered to obtain the segment of the phzH gene and its 5′-end noncoding region of about 2.5 kb long.
2. The recovered segment of the phzH gene and its 5′-end noncoding region was digested by restriction enzymes Xho I and Hind III, and by the action of a ligase, inserted into the corresponding restriction enzyme cutting site of Escherichia coli/Pseudomonas shuttle expression plasmid pBBR1MCS-5 to be under the control of the phage promoter T3 prom, thereby forming the recombination plasmid pBBRphzH which was then transformed into Escherichia coli. On a gentamicin resistant plate, Escherichia coli transformants transformed with pBBRphzH were screened out. Finally, the recombination plasmid was extracted from the Escherichia coli and verified.
3. The competent cells of the derivative strain M18G of the growth-promoting antagonistic strain M18 were prepared, and the aforesaid recombination plasmid pBBRphzH was transformed into the competent cells of M18G which were then cultured at 32° C. for 2 d, and then from which the genetic engineering strain M18G/pBBRphzH for production of phenazine-1-carboxamide was screen out.
The preparation of the competent cells of the derivative strain M18G of the growth-promoting antagonistic strain M18, transformation of the recombination plasmid pBBRphzH into the competent cells of M18G, and screening of the genetic engineering strain M18G/pBBRphzH for production of phenazine-1-carboxamide were all carried out according to the methods as described in p 96-99, Chapter 1 of Molecular Cloning: A Laboratory Manual (3rd edition) edited by J. Sambrook and D. W. Russell and published by Science Press in 2002.
4. The genetic engineering strain M18G/pBBRphzH constructed by the gene engineering technology was inoculated onto a plate with a glycerol culture medium where M18G/pBBRphzH was activated and grew at 30° C. for 20 h; then, the bacterial mass was again streaked onto a plate with a glycerol culture medium and activated at 30° C. for 12 h. Then, the activated M18G/pBBRphzH strain was transferred into a 250 ml triangular flask containing 25 ml of a glycerol culture medium, and shake-cultured on a shake table at 30° C. for 11 h, the rotation speed of the shaking table being 180 rpm. Finally, the strain was transferred into a 500 ml triangular flask containing 65 ml of a bacteriocin-producing culture medium where the strain was enlarged fermentation-cultured for 72 h, the temperature and rotation speed being unchanged, thereby obtaining phenazine-1-carboxamide with a yield of 300 mg/L in the fermentation broth. The content of phenazine-1-carboxylic acid was not detected out.
Specifically, the components of the glycerol culture medium and the bacteriocin-producing culture medium were the same as in Example 1.
With this formula, the yield of phenazine-1-carboxamide was 300 mg/L. The content of phenazine-1-carboxylic acid was zero, indicating that all phenazine-1-carboxylic acid had transformed into phenazine-1-carboxamide. As shown by the detection results, in the engineering strain M18/pBBRphzH, the rate of transformation of phenazine-1-carboxylic acid into phenazine-1-carboxamide was 100%.
Example 5
1. The complete coding region of the phzH gene and its 5′-end noncoding region were amplified by using the same method as in Example 1. The product was detected by 1.0% agarose electrophoresis and recovered to obtain the segment of the phzH gene and its 5′-end noncoding region of about 2.5 kb long.
2. The recovered segment of the phzH gene and its 5′-end noncoding region was digested by restriction enzymes Xho I and Hind III, and by the action of a ligase, inserted into the corresponding restriction enzyme cutting site of Escherichia coli/Pseudomonas shuttle expression plasmid pBBR1MCS-5, so that expression of the phzH gene was under the control of the phage promoter T3 prom, thereby forming the recombination plasmid pBBRphzH which was then transformed into Escherichia coli. On a gentamicin resistant plate, Escherichia coli transformants transformed with pBBRphzH were screened out. Finally, the recombination plasmid was extracted from the Escherichia coli and verified.
3. The competent cells of the Pseudomonas PAO1 strain were prepared, and the aforesaid recombination plasmid pBBRphzH was transformed into the competent cells of PAO1 which were then cultured at 32° C. for 2 d, and then from which the genetic engineering strain PAO1/pBBRphzH for production of phenazine-1-carboxamide was screen out.
The preparation of the competent cells of the Pseudomonas PAO1 strain, transformation of the recombination plasmid pBBRphzH into the competent cells of PAO1, and screening of the genetic engineering strain PAO1/pBBRphzH for production of phenazine-1-carboxamide were all carried out according to the methods as described in p 96-99, Chapter 1 of Molecular Cloning: A Laboratory Manual (3rd edition) edited by J. Sambrook and D. W. Russell and published by Science Press in 2002.
4. The genetic engineering strain PAO1/pBBRphzH constructed by the gene engineering technology was inoculated onto a plate with a glycerol culture medium where M18G/pBBRphzH was activated and grew at 30° C. for 20 h; then, the bacterial mass was again streaked onto a plate with a glycerol culture medium and activated at 30° C. for 12 h. Then, the activated PAO1/pBBRphzH strain was transferred into a 250 ml triangular flask containing 25 ml of a glycerol culture medium, and shake-cultured on a shake table at 30° C. for 11 h, the rotation speed of the shaking table being 180 rpm. Finally, the strain was transferred into a 500 ml triangular flask containing 65 ml of a bacteriocin-producing culture medium where the strain was enlarged fermentation-cultured for 72 h, the temperature and rotation speed being unchanged, thereby obtaining phenazine-1-carboxamide with a yield of 50 mg/L in the fermentation broth. The content of phenazine-1-carboxylic acid was not detected out.
Specifically, the components of the glycerol culture medium and the bacteriocin-producing culture medium were the same as in Example 1.
With this formula, the yield of phenazine-1-carboxamide was 50 mg/L. The content of phenazine-1-carboxylic acid was not detected out, indicating that all phenazine-1-carboxylic acid had transformed into phenazine-1-carboxamide. As shown by the detection results, in the engineering strain PAO1/pBBRphzH, the rate of transformation of phenazine-1-carboxylic acid into phenazine-1-carboxamide was 100%.
Example 6
Detection of Stability in Antifungal Activity of phenazine-1-carboxamide as Fermentation Product
The acidity of a potato glucose culture medium was adjusted with a phosphate-citrate buffer to 4.0, 5.0, 6.0, 7.0, and 8.0 respectively. To the potato glucose culture media with different acidities, a certain amount of each of an M18G/pME6032Phz fermentation broth and an M18G/pBBRphzH fermentation broth was added respectively, thereby obtaining potato glucose culture media with diluted 16 mg/L of phenazine-1-carboxylic acid and phenazine-1-carboxamide respectively having different acidities. The potato glucose culture media were then poured into 9 cm diameter petri dishes to form flat plates, a potato glucose culture medium plate without any fermentation broth was used as a blank control, and each treatment had three repetitions. A rice sheath blight pathogenic hypha mass with a diameter of 8 mm was inoculated at the center of the plate, and incubated in a thermostatic incubator at 28° C. Once the pathogenic hyphae in the blank control grew all over the petri dish, the diameters of the fungal masses inoculated in the potato glucose culture media were respectively measured by using the crossing method. Antifungal rate, that is inhibition rate of fungal growth is calculated as follows: antifungal rate=(1−(D2−Din2)/(Dck2−Din2))×100%, where D represents the average diameter of the hypha mass in the treatment group; Din represents the initial diameter of the hypha mass; Dck represents the average diameter of the hypha mass in the control. The analysis results are shown in FIG. 3, wherein PCA represents phenazine-1-carboxylic acid, and PCN represents phenazine-1-carboxamide. The results show that, under acidities of 4.0, 5.0, 6.0, 7.0, 8.0, the antifungal activities of phenazine-1-carboxylic acid (PCA) against rhizoctonia solani were 95.5%, 82.9%, 67.5%, 15.9%, and 0 respectively; the antifungal activities of phenazine-1-carboxamide (PCN) against rhizoctonia solani were 65%, 69%, 73.6%, 80.1%, and 90% respectively. That is, under pH of 4-8, as compared with phenazine-1-carboxylic acid, the antifungal rate of phenazine-1-carboxamide against rhizoctonia solani was stable; meanwhile, under pH of 7.0, the antifungal activity of phenazine-1-carboxamide against Rhizoctonia solani was 5 times that of phenazine-1-carboxylic acid.
Specifically, the potato glucose culture medium comprised the following components in their respective weight percentages: 20% of potato, 2% of glucose, 1.5% of agar, the balance being water.

## Claims

1. A bioengineering strain for production of microorganism-originated fungicides, wherein the bioengineering strain is obtained by transforming a phzH gene expression plasmid into a strain producing phenazine-1-carboxylic acid, wherein the bioengineering strain produces phenazine-1-carboxamide; the phzH gene expression plasmid is an expression plasmid cloned with a phzH gene fragment; the phzH gene expression plasmid expresses phzH gene in the strain producing phenazine-1-carboxylic acid where the phzH gene is encoded to produce PhzH (glutamine phenazine-1-carboxylic acid amidotransferase), the amino acid sequence of the PhzH is SEQ ID NO:1; the PhzH is a PhzH of Pseudomonas aeruginosa strains PAO1, and the phzH gene segment is a phzH gene segment of Pseudomonas aeruginosa strains PAO1; the nucleotide base sequence of the phzH gene fragment comprises a complete coding region of the phzH gene and a 5′-end noncoding region; the sequence of the 5′-end noncoding region comprises a polynucleotide segment starting from the first nucleotide base to the 683th nucleotide base of SEQ ID NO.2; and the strain producing phenazine-1-carboxylic acid is a growth-promoting antagonistic bacterium M18 or an engineering strain M18G.

2. The bioengineering strain of claim 1, wherein the nucleotide base sequence of the phzH gene segment is SEQ ID NO: 2.

3. The bioengineering strain of claim 1, wherein an expression vector for constructing the phzH gene expression plasmid is an Escherichia coli/Pseudomonas shuttle expression plasmid.

4. The bioengineering strain of claim 3, wherein the Escherichia coli/Pseudomonas shuttle expression plasmid comprises a strong promoter, the phzH gene segment is cloned behind the strong promoter, and expression of the phzH gene segment is controlled by the strong promoter.

5. The bioengineering strain of claim 4, wherein the Escherichia coli/Pseudomonas shuttle expression plasmid includes plasmids of pBBR1MCS series.

6. The bioengineering strain of claim 5, wherein the Escherichia coli/Pseudomonas shuttle expression plasmid is pBBR1MCS-5 and the phzH gene is controlled by a phage promoter T3 of the pBBR1MCS-5 on the premise of ensuring a correct reading frame.

7. The bioengineering strain of claim 1 for use in the production of microorganism-originated fungicides through fermentation.

8. A microorganism-originated fungicide, which is a fermentation broth of the bioengineering strain of claim 1.

9. The microorganism-originated fungicide of claim 8, comprising the main fungicidal active component of the fermentation broth comprises phenazine-1-carboxamide.

10. The microorganism-originated fungicide of claim 9, which is in the fermentation broth, and the content of phenazine-1-carboxamide is 2500-2800 mg/L.

11. The microorganism-originated fungicide of claim 8, comprising the microorganism-originated fungicide which is obtained by fermenting and culturing the bioengineering strain of any one of claims 1, 4-5 and 6 under conditions suitable for expression of PhzH and production of phenazine-1-carboxylic acid.

12. A pesticide composition for prophylaxis and treatment of plant diseases, which comprises a fungicidally effective amount of the microorganism-originated fungicide of claim 8 or a fungicidally active component from the microorganism-originated fungicide of claim 8.