CZ299943B6 - Nucleic acid for preparing manumycin-producing strain - Google Patents

Abstract

A nucleic acid encoding manumycin biosynthesis is introduced into a microorganism in order to induce production of metabolites with C5N unit. A method of colony-type hybridization is used to verify so treated microorganism wherein the method is characterized in that colonies grown onto nylon membranes are subjected to lysis, DNA thereof is then fixed by UV. Subsequently, hybridization with ASU probe and detection of hybridizing clones serve for verification whether the introduction of a biosynthetic cluster of genes have induced manumycin production.

Description

Nucleic acid for the preparation of a manumycin producing strain

Technical field

The invention relates to the microbial production of secondary metabolites with a C5N unit

BACKGROUND OF THE INVENTION ¹⁰

The C5N unit (2-amino-3-hydroxycyclopent-1-enone) is part of some secondary metabolites that are produced by actinomycetes. These include, in particular, manumycin antibiotics (manumycin, asukamycin), which have significant anti-tumor (inhibition of races famesylation) and anti-inflammatory effects (inhibition of ICE). These include, for example, antibiotics from the moenomycin family (an alternative to β-lactam antibiotics, inhibits the transglycosylation of peptidoglycan) and bafilomycins with a broad spectrum of biological activities (inhibition of vacuolar transport using stimulation of bone tissue recovery, suppression of metastasis in Helicobacter pylori infection). These metabolites share only the presence of the C5N unit in their chemical structure, otherwise they fall into completely different groups of antibiotics (polyketides, macrolides, glycopeptides, etc.). The presence of the C5N unit can be advantageously utilized in the isolation of substances containing it: C5N is a UV chromophore with absorption maxima in the region of 250-300 nm. This property is used, for example, in the isolation of moenomycins from Streptomyces bambergtensis and Streptomyces ghanaensis production cultures.

The C5N unit is formed by cyclization of 5-aminolevuolvic acid (ALA). ALA is the first precursor in tetrapyrrol biosynthesis and is therefore a common intermediate metabolite in all living organisms. It can be synthesized by two biosynthetic pathways: the Shemin pathway, of glycine and succinate-CoA, and the C5 pathway, which utilizes glutamic acid modifications activated in the form of glutamyl-tRNA. With a few exceptions, all living organisms synthesize ALA by only one of the above pathways. With the exception of proteobacteria that exclusively use the Shemin pathway, all other prokaryotes, including actinomycetes, synthesize the ALA C5 pathway, that is, glutamic acid. For the asucamycin producer, however, experiments with labeled precursors indicated that C5N is derived from ALA, which is derived from glycine and succinylCOA. Thus, both pathways should be active.

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

SUMMARY OF THE INVENTION

Streptomyces strains that produce secondary metabolites containing the C5N unit encode and utilize both biosynthetic pathways of 5-aminolevulinic acid synthesis. One of them (Shemin) is specifically used for the production of ALA for secondary metabolism, as opposed to its original function in many eukaryotic organisms, where it serves exclusively for the essential synthesis of porphyrins. Thus, the presence of the key enzyme of the Shemin pathway enzyme, 5-aminotevulinic acid synthase (ALAS), is a genetic "label" for all C5N producers in streptomyces.

-1 CZ 299943 B6

The invention provides the full sequence gene sequence of manumycin biosynthesis (sequence id,

No. 7), which was isolated from the strain Streptomyces parvulus TU64. First, total genomic DNA of the Streptomyces parvulus T64 strain was isolated, which was subsequently enzymatically digested into fragments by restriction endonucleases. The fragments were sized electrophoretically and transferred to a membrane. This membrane is immersed in a solution containing a conserved fragment of the hemA-asuA gene that encodes the biosynthesis of secondary metabolites with a C5N unit and is referred to as a probe. The isolated DNA fragments immobilized on the membrane are incubated in solution with the conserved fragment of the hemA-asuA gene by probe, if any of these fragments hybridizes with the probe, it bears the same genetic information and is thus a producer of secondary metabolites with the C5N unit. These fragments are determined using detection solutions. Thus, a gene cluster was found in which the hybridizing fragment was contained, which in this case encodes the entire manumycin synthesis.

is an overview of the drawings

The attached drawing shows examples of C5N unit secondary metabolites produced by actinomycetes. C5N units are circled. Giant. I Manumycin, Fig. 2 Asukamycin, Fig. 3 Reductiomycin, Fig. 4 Moenomycin A.

DETAILED DESCRIPTION OF THE INVENTION

A complete biosynthetic cluster of manumycin and other minor manumycin genes 25 from Streptomyces parvulus Tu64 (SEQ ID NO: 7) was isolated, cloned, and sequenced using the ASU gene probe. By comparing the DNA sequence with the gene bank and analyzing potential protein domains, the likely open reading frames (ORFs) and the predicted functions of their protein products were determined. Furthermore, the likely boundaries of the gene cluster were defined. The entire cluster is present on a 30.7 kbp DNA segment and consists of 28 ORFs designated M1-M28. This cluster of DNA is inserted into a microorganism selected to induce manumycin production. Colony hybridization was used to verify the microorganism thus treated. Colonies grown on nylon membranes were lysed, and their DNA was fixed by UV. Whether insertion of the biosynthetic cluster of genes resulted in manumycin production was verified by hybridization with the ASU probe and detection of hybridizing clones.

_; Industrial applicability

In this way, antibiotics, in this case manumycin, can be obtained.

Claims (2)

5 PATENT CLAIMS Nucleic acid for the preparation of a manumycin-producing strain having the sequence id. No. 7 io The nucleic acid of claim 1, which is inserted into the vector DNA.