CZ299789B6 - Method of detecting microorganisms producing metabolites comprising C5N unit - Google Patents

Abstract

The present invention relates to a method of detecting microorganisms producing secondary metabolites comprising C5N unit by making use of a probe being an isolated DNA fragment, which represents a conservative section of a hem-A-asuA gene for aminolevulinate synthase from Streptomyces nodosus ssp. asukaensis. The inventive method is characterized by searching a section with the same nucleotide sequence in the DNA of the tested strains, isolating DNA of actinomycete, splitting thereof to fragments, dividing the fragments according to their size and transferring them to a membrane. The membrane is then submerged in a solution containing a conservative fragment of the hem-A-asuA gene of the genome DNA actinomycete Streptomyces nodosus ssp. asukaensis, having the sequence ID No. 5, or a part thereof defined by the sequence of the ID No. 6, from which it is transferred after incubation into detection solutions.

Description

Method for screening procaryotic organisms producing metabolites with C5N unit

Technical field

The invention relates to the screening of prokaryotic organisms producing C5N unit metabolites in isolates of soil microorganisms, in particular actinomyces.

BACKGROUND OF THE INVENTION

The C5N unit (2-atino-3-hydroxycyclopent-1-enone) is part of some secondary metabolites. which are produced by bacteria from the actinomyces family. These include, in particular, manumycins and novel antibiotics (manumycin, asukamycin). which have significant anti-tumor (inhibition of farnesylaee race) and anti-inflammatory effects (inhibition of ICE). They also include antibiotics from the group of mocnomycin (an alternative to β-lactam antibiotics, inhibits the transglycosylation of peptidoglycan) and bafdomycins with a broad spectrum of biological activities (inhibition of vacuolar transport using stimulation of bone tissue restoration, suppression of tumor cell metastasis and in Helicobacter pylori infection). These metaboles20 share only the presence of the C5N unit in their chemical structure, otherwise they fall into completely different classes of antibiotics (polyketides, macrolides, glycopeptides, etc.). The presence of a C5N unit can be advantageously used in the isolation of substances containing it: C5N is a UV bromine with absorption maxima in the region of 250 to 300 nm. This property is used, for example, in the isolation of moenomycins from Streptomyces bambergiensis and Streptomyces gbanacnsis production cultures.

The C5N unit is formed by cyclization of 5-aminolevuolic acid (ALA). ALA is the first precursor to biosynthesis in rap years. and is thus a common intermediate metabolite in all living organisms. It can be synthesized by two biosynthetic pathways: the so-called Shemin pathway, zglycine and succinyl-CoA, and the so-called C5 pathway, which utilizes glutamic acid modifications activated in the form of glutamyl-tRNA. With few exceptions, all living organisms synthesize ALA only one of the above pathways. Excluding and proteobaklerii. using exclusively the Shemin pathway, all other prokaryotes, including actinomycetes, synthesize the ALA C5 pathway, i.e. glutamic acid. For the producer of asucamycin, however, experiments with labeled precursors indicated that C'5N was derived from ALA. which is derived from glyeine and succinyl 35 Co A.

In most cases, the genes encoding the individual steps of secondary metabolite biosynthesis are arranged in clusters. Thus, the complete genetic information allowing the formation of the substance is often contained in a single chromosomal locus. Thus, the identification of a single biosyntholic gene allows easy isolation of the entire gene cluster for further analysis and gene manipulation.

identifying a gene or genes specifically associated with the biosynthesis of biologically active substances containing a particular chemical structure would thus allow for targeted search for new actinomyeet production strains. Information about the fact that particular bacterial strains produce antibiotics would be obtained much earlier than cultivation, because a suitable culture medium on which the bacteria would grow and produce the antibiotic must first be created.

SUMMARY OF THE INVENTION

The present invention provides a method for screening prokaryotic organisms producing metabolites with a C5N unit. DNA is isolated from bacteria of the genus actinomyeet and subsequently enzymatically cleaved into fragments by restriction endonucleases. The fragments were sized electrophoretically and transferred to a membrane. This membrane is immersed in a solution that

- 1 C7. 299789 Bo contains a conserved fragment of the hemA-asuA gene from the active DNA of Streptotnyces nodosus spp. asukaensis having the sequence of SEQ ID NO.5, or a portion thereof defined by SEQ ID NO.6. and is referred to as a probe. The isolated DNA fragments immobilized on the membrane are incubated in solution with a conserved fragment of the hemA-asuA gene by probe if any of these fragments hybridizes with the probe, carrying identical genetic information and thus producing secondary metabolites with a C5N unit. These fragments are determined using detection solutions.

The portion defined by SEQ ID NO: 6 is obtained from a conserved fragment of the hemA-asuA gene by synthesis using a polymerase chain reaction (PRC) and a set of oligonucleotides having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 1. 3 and SEQ ID NO: 4. or by restriction endonuclease (SacI) cleavage.

The invention thus provides a very efficient, i.e. fast and inexpensive, method for screening for specific microorganisms, in particular Streptomyces bacteria. producing secondary metabolites with a C5N unit, in particular manumycin antibiotics, from a large number of samples, for example by colony hybridization or by automated hybridization or PCR scanning.

The hemA-asuA gene was isolated from S. nodosus ssp. which produces asukamyein.

First, a PCR method was used for its isolation - based on the comparison of the conjugate and fairy regions of several known ammulevulinate synthase genes, degenerate primers (PCR loops) of ASU-F and ASU-R were designed to amplify a portion of hemA-asuA. Degenerate primers ASIJ-F and ASU-R were used for amplification:

1: 1 primer mix ASU-F 1 5'-GTSTGGTGYTCSAACGACTACCT-3 '(SEQ ID NO: 1) and

2? ASU-F2 5'-GTSTGG fGYRCSAACGACTACCT-3 '(SEQ ID NO: 2), and

1: 1 mixture of ASU-RI 5'-CCSAAGTAGAAGWGSTGS1 $ SGAS-3 '(SEQ ID NO.3) and ASU-R2 5'-CSAAGTAGAAGTSSTGSISSGAS-3' (SEQ ID NO.4)).

Conventional and well-known symbols are used to denote nucleotides, wherein 1 is inosine, Y is C or T. W is A or T, R is G or A and S is G or C.

The PCR fragment obtained was cloned and its sequence determined (SEQ ID NO: 5). This fragment was further used as a gene probe for hybridization with restriction fragments of S. nodosus chromosomal DNA. The I hybridizing BamHI fragments (ca. 8 kbp) and PstI (7 kbp) were cloned and sequenced (plasmid clones pMAPO1 and pMPA03). Their analysis by BFAST confirmed the presence of the complete hemA-asuA gene and showed the presence of probable secondary metabolism genes in its vicinity.

To demonstrate that the functionality of the hemA asuA gene is required for the production of asucamycin, a mutant strain of S. nodosus hemA asuA; wherein the essential part of the gene is replaced by the apramycin resensin gene. The mutant strain did not differ from the wild strain in its growth characteristics, but its production of asucamycin and other pathway products was completely stopped. Conversely, intermediates lacking the C5N unit accumulated here. Thus, it has been shown that the enzyme responsible for catalysing the first step of C5N biosynthesis (i.e. 5-aminolevulinic acid formation for secondary metabolism purposes) is the 5-aminolevulinic acid synthase, encoded by the asucamycin producer. Streptomyces nodosus var. asukaensis. the hemA-asuA gene. Mere addition of ALA to the culture medium did not restore the mutant asukamycin synthesis.

Part of the original PCR product was used as an ASU gene probe (SEQ ID NO: 6) for the detection of homologous genes in the chromosomal DNA of selected actinomyeet strains by hybridization. This set included some known producers of sC5N: S. nodosus var. asukanesis (asukamyein). S. parvulus Tu64 (manumycin A), S. xanthochromogenus AM6201 (rcductiomycin) and S. bambergiensis DSM40590 and S.ghanaensis DSM40746 (both phenoenomycin)

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AND); and 10 randomly selected known strains from the laboratory collection that do not produce C5N substances (eg S. eoelicolor A3 (2), S. lividans TK24, S. griseus HH1, etc.). Hybridization with the GTR probe for the gene encoding glutamyl tRNA reductase was used as a control. It is actinomycet 'house-keeping' gene and encodes one step of ALA C5 biosynthesis pathway. All of the chromosomal DNA tested hybridized with the GRT probe, but only the DNA of the producer

C5N compounds showed hybridization with soda ASU. These unambiguous results confirmed that the presence of the hemA-asuA gene in the streptomycetes genome is specifically linked to the ability to produce secondary metabolites with the C5N unit and can be used for genetic screening of new aclinomycct isolates in search for producers of new C5N agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are examples of secondary metabolites with a C5N unit produced by actinomyces. C5N units are circled. Giant. 1 Manumycin, Fig. 2 Asukamycin, Fig. 3 Reductiomycin, Fig. 4 Moenomycin A.

DETAILED DESCRIPTION OF THE INVENTION

Soil isolates of new actinomycetes strains were cultured on a rotary shaker in liquid GYM medium at 28 ° C and 200 rpm. Total genomic DNA was isolated from 2-6 ml submerged culture. Two pg of this DNA were digested with BamIII or PstI for 2-3 hours. The restriction fragments were separated by standard agarose electrophoresis in a 1% gel stained with ethidium bromide, and the DNA fragments were blotted onto a nylon membrane. Transferred

DNA was immobilized on the membrane by UV. hybridized with the ASU probe and hybridizing fragments were detected. A membrane with the same DNA samples was also hybridized with the GTR probe. where clear hybridization served as a control of the integrity of the isolated DNA, its good restriction endonuclease digestion, transfer of DNA to the membrane, and proper hybridization. The membranes always contained, as a positive control, the DNA of S. parvulus Tu64 strain (manumycin producer). which hybridizes to both probes.

Industrial Applicability 35

A suitable bacterial strain can be found in a simple manner. which, as its secondary metabolite, produces biologically active substances - antibiotics. By submerged culture of this strain, antibiotics can be produced commercially for the pharmaceutical market.

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Sequence list

SEQ ID NO. 5 <210> 5 <211> 693 <212> DNA <213> synthetic <220>

<221> PC R product <222> (1) ... (693) <223> encodes the operative region of the hemA-asuA gene from Streptonivces nodosus ssp. Asukaensis <400> 5

CZ 299789 Bo

gtctggtgca gcaacgacta cctgggcatg ggccagaacc cgttcgtgct ggaagccgtg 60 aagaacgccg tcgacgcctt cggcgccgga tccggcggct cccgcaacat cggtggcacc 120 aaccactacc acgtcctgct ggagaacgag ctggcggcgc tgcacggcaa ggaggaggcg 180 ctgatcttcc cctccggctt cacggccaac gacggcgcgc tgaccgtgct ggcgggccgg 240 gcgcccggca ccctcgtctt ctccgacgag ctgaaccatg cctcgatcat cgacgggctg 300 cgccaclccg gggccgagaa gcggatcttc cgccacaacg acatggcgca cctggaggaa 360 ctgctggccg ccgcggaccc ggagcggccc aagctgatcg tgctggagtc ggtgtactcg 420 atgtccggcg acatcgcgcc gctcgccgag accgccgccc tggcccgccg tcacggggcg 480 accaccttca tcgacgaggt gcacgcggtc ggcatgtacg gcccgcaggg tgcgggcatc 540 gccgcccgcg aaggcatcgc cqacgagttc accgtggtca tgggcaccct ggccaagggc 600 ttcggtaccg ccggcggcta catcgccggt cccgccgcgc tgatcgacgc cgtccgcaac 660 ttctcccgcg getteatett caccacctcc atc 693

SEQ ID NO. 6 <210> 6 <211> 3OI <212> DNA <213> Synthetic <220>

id <221> SacII fragment from PCR fragment SEQ. ídID, No <5 <222> (1) ... (301) <223> fragment from the conjugate region of the hemA asn A gene from Sireptomyces nodosus ssp. asukaensis i? <400> 6 ccgcgggaga agttgcggac ggcgtcgatc agcgcggcgg gaccggcgat gtagccgccg 60 gcggtaccga agcccttggc cagggtgccc atgaccacgg tgaactcgtc ggcgatgcct 120 tcgcgggcgg cgatgcccgc accctgcggg ccgtacatgc cgaccgcgtg cacctcgtcg 180 atgaaggtgg tcgccccgtg acggcgggcc agggcggcgg tctcggcgag cggcgcgatg 240 tcgccggaca tcgagtacac cgactccagc acgatcagct tgggccgctc cgggtccgcg 300 Q 301

Claims (4)

A method for screening prokaryotic organisms producing metabolites with a C5N unit, 25 comprising isolating DNA from actinomycetes, which is cleaved into fragments, fragmented according to size and transferred to a membrane, characterized in that the membrane is immersed in a solution containing a conservative a fragment of the hemA-asuA gene from the genomic DNA of the actinomycetes Strcptomyces nodosus ssp. asukaensis having the sequence of SEQ ID NO: 5. or a portion thereof, defined by SEQ ID NO: 6, from which it is transferred to detection solutions after incubation. Method according to claim 1, characterized in that the part defined by SEQ ID NO: 6 is obtained from a conserved fragment of the hcmA-asuA gene by synthesis using a polymerase chain -4C7. 299789 B6 reaction (PCR) and a set of oligonucleotides having the sequence of SEQ ID NO: 1. SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. The method of claim 1, wherein the portion defined by SEQ ID NO: 6 is 5 is derived from a conserved fragment of the heniA-asuA gene by restriction endonuclease digestion (SacII). The method according to claim 1 or 2 or 3, characterized in that the sc fragments are inserted into a vector, which is inserted into the genetic information of Eseheriehia coli, the cells of which are grown into colonies which subsequently lyse and niobialize on the membrane. which is immersed in a solution containing a conserved fragment of the hemA-asuA gene from the genomic DNA of the actinomyeet of Streplomyces nodosus ssp. asukaensis having the sequence of SEQ ID NO.5 or a portion thereof defined by SEQ ID NO.6 from which it is transferred after incubation into detection solutions.