Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-5-aza-bicyclo
Reexamination Certificate
1999-06-15
2001-01-30
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-5-aza-bicyclo
C435S043000, C435S044000, C435S048000, C435S071100, C435S071200, C435S071300, C435S183000, C435S252300, C435S252310, C435S252330, C435S252350, C435S254110, C435S254500, C435S320100, C435S254300, C536S023200, C536S023100, C536S023700, C530S350000
Reexamination Certificate
active
06180360
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to biosynthesis of &bgr;-lactam antibiotics. More specifically, the invention relates to in vivo and in vitro processes of producing &bgr;-lactam antibiotics.
Also contemplated is a novel enzyme which advantageously can be used in the &bgr;-lactam antibiotic biosynthesis. Further the invention relates to a DNA construct encoding said novel enzyme, a recombinant vector or transformation vehicle comprising said DNA construct, and finally a cell comprising said DNA construct or recombinant vector.
BACKGROUND OF THE INVENTION
Biosynthetic pathway of penicillin
The first step in the biosynthesis of penicillin involves the formation of the tripeptide &dgr;-(L-&agr;-aminoadipyl)-L-cysteinyl-D-valine (ACV) from L-&agr;-aminoadipic acid, L-cysteine and L-valine (Fawcett et al., Biochem. J., 157, p. 651-660, 1976). The reaction is catalyzed by a multifunctional enzyme &dgr;-(L-&agr;-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase) with ATP and Mg
2+
as co-factors (Banko et al., J. Am. Chem. Soc., 109, p. 2858-2860, 1987).
ACV synthetase (ACVS) has been purified from
Aspergillus nidulans
(Van Liempt et al., J. Biol. Chem., 264, p. 3680-3684, 1989),
Cephalosporium acremonium
(Baldwin et al., J. Antibiot., 43, p. 1055-1057, 1990) and
Streptomyces clavuligerus
(Jensen et al., J. Bacteriol., 172, p. 7269-7271, 1990, and Zhang et al., Biotechnol. Lett., 12, p. 649-654, 1990). The purification of ACV synthetase from
Penicillium chrysogenum
has not been published. However, ACV synthetase from
P. chrysogenum
has been cloned by Diez et al. (J. Biol. Chem., 265, p. 16358-16365, 1990).
The linear tripeptide, ACV, is converted to isopenicillin N (IPN) in the presence of isopenicillin N synthase (also referred to as cyclase or isopenicillin N synthetase (IPNS)), ferrous ions, oxygen and an electron donor (lag ascorbate). Isopenicillin N synthase was first isolated from
P. chrysogenum
by Ramos et al. (Antimicrobial Agents and Chemotherapy, 27, p. 380-387, 1985) and the isopenicillin N synthase structural gene from
P. chrysogenum
cloned by Carr et al. (Gene, 48, p. 257-266, 1986).
These first two steps in the biosynthesis of penicillins are common in penicillin and cephalosporin producing fungi and bacteria.
In some fungi, for example in
P. chrysogenum
and in
A. nidulans
, the &agr;-aminoadipyl side chain of isopenicillin N can be replaced by other side chains of intracellular origin or exogenously supplied. The exchange is catalyzed by an acyltransferase (referred to as acyl-coenzyme A: isopenicillin N acyltransferase or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase). It is still unclear whether this conversion proceeds in vivo by a two-step reaction in which first the L-&agr;-aminoadipyl side chain is removed to yield 6-aminopenicillanic acid (6-APA) followed by the acylation step, or the conversion is a direct exchange of the side chains. Purified acyltransferase from
P. chrysogenum
has both an isopenicillin N-amidohydrolase activity and an acyl-coenzyme A:6-aminopenicillanic acid acyltransferase activity Alvarez et al. Antimicrobial Agents and Chemotherapy, 31, p. 1675-1682, 1987).
The genes coding for ACV synthetase (pcbAB), isopenicillin N synthase (pcbC) and acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (penDE) are found in the same cluster in
P. chrysogenum
and
A. nidulans
(Diez et al., J. Biol. Chem., 265, p. 16358-16365, 1990, and Smith et al., Bio/Technology, a, p. 39-41, 1990).
Amplification of the pcbC-penDE gene cluster of
P. chrysogenum
Wis 54-1255, coding for isopenicillin N synthase (IPNS) and acyltransferase (AT), respectively, led to as much as a 40% improvement in production yields (Veenstra et al., J. Biotechnol., 17, p. 81-90, 1991). Increased antibiotic yields were also reported in
A. nidulans
transformants containing multiple copies of pcbAB (coding for ACV synthetase (ACVS)) and pcbC genes (coding for isopenicillin N synthetase (INPS)) (McCabe et al., J. Biotechnol., 17, p. 91-97, 1991).
EP 200425 (Eli Lilly) discloses vectors encoding isopenicillin N synthetase (IPNS). The vectors permit high level expression of IPNS in
C. acremonium
and
E. coli
. According to the disclosure the Cephalosporium vectors are useful for strain improvement, to increase efficiency and yield in fermentations for the production of penicillin and cephalosporin antibiotics. The vectors may also be modified to give vectors for increasing the production yields and efficiency of
P. chrysogenum, Streptomyces clavuligerus
etc. in fermentations.
EP 357119 (Gist Brocades) discloses the clustered antibiotic biosynthetic genes encoding IPNS, AT and ACVS and are advantageously employed for improvement of production of the antibiotic in microorganisms and for the isolation of other genes involved in the biosynthesis of the antibiotic. The invention is exemplified with improved production of penicillin in
P. chrysogenum
, with the isolation of another clustered biosynthetic gene(s) and with the expression of clustered penicillin biosynthetic genes in
Acremonium chrysogenum.
Activation of side chain
In order to replace the &agr;-aminoadipic acid side chain in the acyltransferase catalyzed reaction, the carboxylic acid group of the new side chain has to be activated. This activation is one of the least well understood parts of the biosynthesis of penicillins. Two theories have been proposed.
The most widely accepted theory is that the enzyme catalyses the esterification of carboxylic acids into coenzyme A thioesters by a two-step mechanism that proceeds through the pyrophosphorolysis of ATP (adenosine triphosphate), in the presence of Mg
2+
. Firstly, the carboxylic acid (the new side chain), ATP and the enzyme forms a complex, leading to an acyl-AMP-enzyme complex. Secondly, this complex reacts with coenzyme A to liberate acyl-coenzyme A and AMP (adenosine monophosphate).
The other theory is based on the formation of an acyl-S-glutathione intermediate, which may be transformed to the corresponding acyl-coenzyme A ester (Ferrero et al., J. Antibiot., 43, p. 684-91, 1990.
A phenacyl:coenzyme A ligase from
P. chrysogenum
able to catalyze the synthesis of phenoxyacetyl-coenzyme A and phenylacetyl-coenzyme A in the presence of ATP, Mg
2+
, coenzyme A and phenoxy-acetic acid or phenylacetic acid has been described by Brunner, Rohr and Zinner (Hoppe-Seyler's Z. Physiol. Chem., 349, p. 95-103, 1968), Brunner and Rohr (Methods Enzymol., 43, p. 476-481, 1975; Kogekar and Deshpande, Ind. J. Biochem. Biophys., 19, p. 257-261, 1982, and by Kurzatkowski, Med. Dosw. Mikrobiol., 33, p. 15-29, 1981). According to Brunner et al., the ligase shows similar degrees of activity towards phenylacetic acid, phenoxyacetic acid and acetic acid. However, the enzyme was never purified to homogeneity.
Martinez-Blanco et al. (J. Biol. Chem., 267, p. 5474-5481, 1992) have described an acetyl-coenzyme A synthetase from
P. chrysogenum
Wis 54-1255 which not only accepts acetic acid but also phenylacetic acid as substrates in the synthesis of the corresponding acyl-coenzyme A esters just like the ligase described by Brunner et al. However, the activity towards phenoxyacetic acid is not described by Martinez-Blanco et al. According to Martinez-Blanco et al., the acetyl-coenzyme A synthetase is a homo-dimer (&agr;
2
) having a molecular weight of 139,000 Dalton as determined by gel filtration and of 70,000 Dalton as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and an isoelectric point: between pH 5.6 and 6.0.
The gene coding for the acetyl-coenzyme A synthetase of Martinez-Blanco et al. has been characterized by Martinez-Blanco et al. (Gene, 130, p. 265-270, 1993). The gene which was designated acuA contains five introns and codes for a polypeptide of 669 amino acids. This polypeptide has a molecular weight of 74,287.
Gouka et al. (Appl. Microbiol. Biotechnol., 38, p. 514-519, 1993) and Van Hartingsveldt et al. (WO 92/07079) have described the isolation and sequence of
Kaasgaard Svend
Kristiansen Klaus
Mølgaard Henrik
Gist-Brocades
Hutson Richard
McDonnell & Boehnen Hulbert & Berghoff
Prouty Rebecca E.
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