Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-4-aza-bicyclo
Patent
1997-03-05
1999-03-16
Jacobson, Dian C.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-4-aza-bicyclo
4352523, 43525411, 4353201, 435476, 536 232, 536 241, C12N 115, C12N 121, C12N 1563, C12P 3700
Patent
active
058828834
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/DK95/00386 filed 28 Sept. 1995 and claims priority under 35 U.S.C. 119 to Danish application 1118/94 filed 28 Sept. 1994, the contents of which applications are fully incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to processes for the production of secondary metabolites in high yields by use of modified microorganisms. The invention further relates to processes for producing the modified microorganisms, DNA constructs and vectors for use in such processes and the modified microorganisms. Lastly the invention relates to secondary metabolites produced by the first mentioned methods.
BACKGROUND OF THE INVENTION
The biochemical pathways of microorganisms can be classified as being part of either primary or secondary metabolism. The pathways of primary metabolism are involved in the catabolism of molecules for energy production or in the synthesis of the building blocks of the cells. Most of these processes are common for all microorganisms. The secondary metabolism is usually anabolic and leads to compounds with no obvious function for the cell.
Among secondary metabolites .beta.-lactam antibiotics are a large family produced in nature by microorganisms. The most important class of .beta.-lactam antibiotics both clinically and economically are the penicillins and the cephalosporins. Their biosynthesis occur via a complex pathway of enzymatic steps.
The unravelling of this pathway has been the subject of many studies during the last few decades. The first two steps in the biosynthetic pathways of the penicillin and the cephalosporin classes of .beta.-lactam antibiotics are identical. Thereafter the biosynthetic pathways to the penicillins and cephalosporins diverge.
The pathway to the important penicillin species penicillin V is sketched below.
Aminoadipic acid+cysteine+valine.fwdarw.ACV-tripeptide.fwdarw.isopenicillin N.fwdarw.penicillin V
The first step is a condensation of L-.alpha.-aminoadipic acid (an intermediate in the lysine biosynthetic pathway in fungi or a degradation product of lysine in bacteria), L-cysteine and L-valine. In cephamycin-producing Actinomycetes, lysine (an amino acid belonging to the so-called aspartate family) is synthesized by the dihydrodipicolinate pathway, which does not include .alpha.-aminoadipic acid as an intermediate. In these organisms the precursor is formed by catabolism of lysine by the action of lysine-6-aminotransferase.
In the second step, ACV is oxidatively cyclized by removal of four hydrogen atoms to form the bicyclic penam nucleus (a .beta.-lactam fused to a thiazolidine ring) of isopenicillin N which is present in all penicillins. From here the pathway diverges to penicillins in Penicillium chrysogenum and Aspergillus nidulans and to cephalosporins and cephamycins in various molds and Actinomycetes. Cephalosporins and cephamycins (7-.alpha.-methoxycephalosporins) contain the cephem bicyclic ring system (a .beta.-lactam fused to a dihydrothiazine ring).
Formation of the ACV-tripeptide is carried out by the enzyme .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS).
The enzyme catalyzing the second step in the penicillin, cephalosporin and cephamycin biosynthesis is isopencillin N synthase (IPNS or cyclase). IPNS is stimulated by ferrous ions and ascorbate, and requires a reduced environment. As the .beta.-lactam ring is formed during cyclization, isopenicillin N (IPN) is the first compound produced in the pathway with antibiotic activity.
IPNS has been purified from a wide variety of .beta.-lactam producing organisms including Streptomyces clavuligerus, Streptomyces lactamdurans, Penicillium chrysogenum and Cephalosporium acremonium.
The final step in the penicillin biosynthesis is catalyzed by acyl-CoA:6-aminopenicillanic acid transferase (AT), which has been purified from organisms as e.g. Penicillium chrysogenum and Aspergillus nidulans.
Most of the enzymes involved in the biosynthesis of .bet
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The Merck Index, Eleventh Edition, "Penicillin G Potassium", p. 1123.
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Egel-Mitani Michi
Kaasgaard Svend
Kristiansen Klaus Nyegaard
M.o slashed.llgaard Henrik
Gregg, Esq. Valeta
Jacobson Dian C.
Novo Nordisk A S
Zelson Esq. Steve T.
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