Process for increasing the production of penicillin G...

Details Process for increasing the production of penicillin G... Process for increasing the production of penicillin G...

1998-10-28

2001-06-26

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S043000, C435S183000, C435S320100, C536S023200, C530S350000

Reexamination Certificate

active

06251655

ABSTRACT:

FIELD OF THE INVENTION
This invention describes a new process for obtaining strains of
Penicillium chrysogenum
with greater penicillin G production capacity, by the introduction and expression in the control strain of an exogenous gene which codes for the enzyme phenylacetyl-CoA ligase and which originates from the bacterium
Pseudomonas putida.
PRIOR ART
The pathway for biosynthesis of benzylpenicillin (penicillin G) in
Penicillium chrysogenum
is a linear and branched metabolic pathway which leads on one side to the amino acid
L
-lysine and on the other to the said antibiotic (see FIG.
1
).
The penicillins-specific branch begins with the non-ribosomal condensation of three amino acids (
L
-&agr;-aminoadipic,
L
-cysteine and
L
-valine), giving rise to a linear tripeptide (
L
-&agr;-aminoadipyl-
L
-cysteinyl-
D
-valine), also called ACV, which lacks antibacterial activity (Ref. 1). The enzyme which catalyses this conversion is
L
-&agr;-aminoadipyl-
L
-cysteinyl-
D
-valine synthetase, which in abbreviated form is called ACVS (Ref. 2). In a subsequent step the tripeptide ACV is converted to isopenicillin N (IPN) by the cyclization of the
L
-cysteine and
D
-valine residues (Ref. 3). This reaction leads to the synthesis of a molecule which has two rings, &bgr;-lactam and thiazole, and in which the remainder of the
L
-&agr;-aminoadipic acid remains as a side chain (
FIG. 1
, Ref. 3). IPN is the first molecule in the pathway which has antibacterial activity, although its potency against G-organisms is poor. The enzyme responsible for synthesis of this compound is isopenicillin N synthetase (IPNS) (Ref. 3). In a subsequent stage (see
FIG. 1
)
Penicillium chrysogenum
replaces the remainder of the
L
-&agr;-aminoadipic acid present in the IPN with phenylacetic acid, giving rise to a molecule which keeps the &bgr;-lactam and thiazole rings but which now has phenylacetic acid as a side chain. This penicillin, called penicillin G or benzylpenicillin, has a greater potency than IPN and a much broader antibacterial spectrum.
The final stage of penicillin G biosynthesis requires at least three different enzymatic reactions. First, the phenylacetic acid has to be incorporated from the culture medium into the cell. This step is catalyzes by a specific transport system referred to by the abbreviation PTS (
FIG. 1
) (Ref. 4). Next, the phenylacetic acid is activated to phenylacetyl-CoA (PA-CoA) by a mechanism, not well known, which appears to require the involvement of a phenylacetyl-CoA-ligase (PCL). Finally, the PA-COA is used by the enzyme acyl-CoA:6-aminopenicillanic acid (isopenicillin N) acyltransferase (AT) (Ref. 5), in such a way that this protein catalyzes the acylation of the 6-amino group of the 6-aminopenicillanic acid (6-APA) or else the interchange between the remainder of the
L
-&agr;-aminoadipic acid present in the IPN molecule and phenylacetyl-CoA, releasing the products penicillin G and CoA in the first case (when the substrates are 6-APA and phenylacetyl-CoA) and penicillin G, CoA and
L
-&agr;-aminoadipic acid in the second case (when the substrates are IPN and PA-COA) (Ref. 6).
The biochemical and genetic studies carried out to date (Ref. 5) have allowed all he enzymes of the penicillin G specific biosynthetic pathway to be identified and their genes to be characterized, with the exception of the enzyme phenylacetyl-CoA ligase, which it has not been possible to purify and the gene of which is unknown at the moment. In addition, the amounts of the different biosynthetic enzymes (ACVS, IPNS and AT) detected in different strains of
Penicillium chrysogenum
(both in those whose production of penicillin G is low and in other strains used industrially) are sufficiently high to eliminate the possibility of any of them being considered a limiting stage in the biosynthesis of penicillin G (Ref. 7-8). For this reason a study was commenced of the enzyme phenylacetyl-CoA ligase (PCL), the only enzyme in the pathway for which the sequence is not fully known. The absence of detectable amounts of enzyme in all the strains of
Penicillium chrysogenum
studied means that different microorganisms have to be selected for their ability to grow in a medium of defined composition (minimal medium, MM) containing phenylacetic acid (PA) as the sole carbon source (Ref. 9), and also that the existence of phenylacetyl-CoA ligase activity has to be assessed in all the selected strains. Of all the microorganisms isolated, a strain of
Pseudomonas putida
U was selected which breaks down phenylacetic acid aerobically by means of an undescribed degradation pathway involving a new enzyme: phenylacetyl-CoA ligase (EC 6.2.1.30). This enzyme was purified to homogeneity and characterized biochemically (Ref. 9). The
Pseudomonas putida
U enzyme, which we will hereinafter call PLC, presents some optimal physicochemical conditions which are very similar to the IPNS and AT of
Penicillium chrysogenum,
and so it was thought that the three enzymes could work together in vitro. It was shown that the PCL of
Pseudomonas putida
U and the IPNS and AT of
Penicillium chrysogenum
could be linked in vitro and used in this form for the production of both penicillin G and other penicillins in the laboratory (Ref. 10). These results, which are described in Spanish Patents Nos. P8902421 and 2016476 A6, allowed the possibility to be suggested that the PCL of
Pseudomonas putida
U might be expressed in
Penicillium chrysogenum
and that, if this enzyme was a limiting stage in the biosynthetic pathway, greater production of penicillin G might be achieved.
DESCRIPTION OF THE INVENTION
1. Isolation of the gene which codes for the enzyme phenylacetyl-CoA ligase in
Pseudomonas putida
U
The strain of
Pseudomonas putida
U, which had phenylacetyl-CoA ligase activity when grown in the MM described in Ref. 9, was mutated by the insertion of the transposon Tn5 (Ref. 11), as is detailed in the protocol shown in FIG.
2
. The strains which were unable to break down phenylacetic acid were selected, which suggested that the insertion had occurred in one of the genes, or intergenic regions, corresponding to the catabolic pathway of this aromatic compound. In all the mutants PCL activity was assayed as described in Spanish Patent P8902421 and in the corresponding publication (Ref. 9). For this purpose the various mutants were grown in the same MM, but it now contained, as carbon sources, 4-hydroxyphenylacetic acid (4-OHPA), which does not induce PCL, and phenylacetic acid (PA), which, although it cannot be broken down, could induce PCL (Ref. 12). In this MM the 4-OHPA is used by the bacteria to sustain cell growth whereas the PA acts as an inducer of the enzyme phenylacetyl-CoA ligase.
By this simple procedure the various mutants were characterized in such a way that two groups could be established:
a) those which possessed functional PCL (called PCL+) and in which the transposon Tn5 had thus inserted itself into a gene on the pathway (or into an intergenic region) after the gene coding for PCL, and
b) the others in which this activity could not be detected (called PCL−).
The absence of PCL in this second group of mutants could be due to two reasons:
1) it could be due to the fact that the transposon had inserted itself in front of the gene coding for the ligase (pcl) (if, as was suspected, all the catabolic pathway responsible for the breakdown of PA is under the control of one promoter), or it could be due to the fact that
2) the Tn5 had incorporated itself into the pcl gene itself, or into a regulator gene or sequence.
From one of the mutants in which no PCL activity was detected, called E
1
, the insertion of Tn5 was identified by the use of oligonucleotide sequences which were exactly the same as the ends of Tn5 (5′ D 3′: ACT TGT GTA TAA GAG TCA G SEQ ID NO:13) and which had been radioactively labelled. The zone of the E
1
mutant genome linked to the transposon was cloned in the plasmid pUC 18 and the
Escherichia coli
strain D5&agr;′ was transformed in accordance with the conventiona

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