Recombinant DNA expression vectors and DNA compounds that encode isopenicillin N synthetase from aspergillus nidulans

DNA compounds and recombinant DNA expression vectors that encode and drive expression in recombinant host cells of the isopenicillin N synthetase activity of Aspergillus nidulans are useful to produce isopenicillin N synthetase and to improve the yield of .beta.-lactam-containing antibiotics from antibiotic-producing organisms. The isopenicillin N synthetase gene of A. nidulans can be isolated from plasmid pOGOO4, available from the Northern Regional Research Center under the accession number NRRL B-18171.

SUMMARY OF THE INVENTION 
The present invention comprises a DNA sequence that encodes the 
isopenicillin N synthetase activity of Aspergillus nidulans. Isopenicillin 
N synthetase catalyzes the reaction in which isopenicillin N is formed 
from .delta.-(-.alpha.-aminoadipyl)-L-cysteinyl-D-valine. This reaction is 
a critical step in the biosynthesis of important antibiotics such as 
penicillins from Aspergillus nidulans, Penicillium chrysogenum, 
Cephalosporium acremonium, and Streptomyces clavuligerus; cephalosporins 
from C. acremonium; and 7a-methoxycephalosporins from S. clavuligerus. 
The novel DNA sequence that encodes the isopenicillin N synthetase activity 
was isolated from Aspergillus nidulans and is useful to construct 
recombinant DNA expression vectors that drive expression of the activity. 
The present invention includes vectors that drive high-level expression of 
the isopenicillin N synthetase activity in E. coli. 
The E. coli-produced isopenicillin N synthetase activity catalyses the 
reaction that forms isopenicillin N from 
.delta.-(-.alpha.-aminoadipyl)-L-cysteinyl-D-valine. Crude cell extracts 
from E. coli transformed with the E. coli vectors of the present invention 
exhibit isopenicillin N synthetase activity without any prior activation 
treatment. The E. coli vectors of the present invention thus provide an 
efficient means for obtaining large amounts of active isopenicillin N 
synthetase. Isopenicillin N synthetase is useful, not only for the 
production of isopenicillin N, but also for the condensation of 
tripeptides other than .delta.-(L-.alpha.- 
aminoadipyl)-L-cysteinyl-D-valine to form novel antibiotics. 
The DNA compounds encoding isopenicillin N synthetase are readily modified 
to construct expression vectors that increase the efficiency and yield of 
antibiotic fermentations involving other organisms, such as Aspergillus 
nidulans, Cephalosporium acremonium, Penicillium chrysogenum, and 
Streptomyces clavuligerus. Although the isopenicillin N 
synthetase-encoding DNA of the present invention was isolated from 
Aspergillus nidulans, the present DNA compounds can be used to construct 
vectors that drive expression of isopenicillin N synthetase activity in a 
wide variety of host cells, as the E. coli vectors of the present 
invention illustrate. All organisms that produce penicillins and 
cephalo-sporins utilize the common precursors 
.delta.-(L-.alpha.-amino-adipyl)-L-cysteinyl-D-valine and isopenicillin N. 
Therefore, the isopenicillin N synthetase-encoding DNA compounds of the 
present invention can be used to produce vectors useful for improving 
efficiency and yield of fermentations involving penicillin and 
cephalosporin antibiotic-producing organisms of all genera. 
The isopenicillin N synthetase-encoding DNA compounds of the present 
invention were derived from Aspergillus nidulans genomic DNA and were 
isolated in conjunction with the transcription and translation activating 
sequence that controls the expression of the isopenicillin N 
synthetase-encoding genomic DNA. The present invention comprises this 
novel transcription and translation activating sequence, which can be used 
to drive expression of genes in A. nidulans and related organisms. 
The present invention also comprises the regulatory signals of the 
isopenicillin N synthetase gene that are located at the 3' end of the 
coding strand of the coding region of the gene. These 3' regulatory 
sequences encode the transcription termination and mRNA polyadenylation 
and processing signals of the Aspergillus nidulans isopenicillin N 
synthetase gene. The presence of these signals in the proper position (at 
the 3' end of the coding strand of the coding region of the gene to be 
expressed) in an expression vector enhances expression of the product 
encoded by the vector. 
The following section provides a more detailed description of the present 
invention. For purposes of clarity and as an aid in understanding the 
invention, as disclosed and claimed herein, the following items are 
defined below. 
aIPNS - isopenicillin N synthetase-encoding DNA of Aspergillus nidulans. 
Antibiotic - a substance produced by a microorganism that, either naturally 
or with limited chemical modification, will inhibit the growth of or kill 
another microorganism or eukaryotic cell. 
Antibiotic Biosynthetic Gene - a DNA segment that encodes an activity that 
is necessary for a reaction in the process of converting primary 
metabolites into antibiotics. 
Antibiotic-Producing Organism - any organism, including, but not limited 
to, Aspergillus, Streptomyces, Bacillus, Monospora, Cephalosporium, 
Penicillium, and Nocardia, that either produces an antibiotic or contains 
genes that, if expressed, would produce an antibiotic. 
Antibiotic Resistance-Conferring Gene - a DNA segment that encodes an 
activity that confers resistance to an antibiotic. 
ApR - the ampicillin resistance-conferring gene. 
Asp DNA - DNA from Aspergillus nidulans. 
cI857 - a temperature sensitive mutant allele of the cI repressor gene of 
bacteriophage lambda. 
Cloning - the process of incorporating a segment of DNA into a recombinant 
DNA cloning vector. 
cos - phage .lambda. cohesive end sequences. 
Genomic Library - a set of recombinant DNA cloning vectors into which 
segments of DNA, which substantially represent the entire genome of a 
particular organism, have been cloned. 
HmR - the hygromycin B resistance-conferring gene. 
Hybridization - the process of annealing two homologous single-stranded DNA 
molecules to form a double-stranded DNA molecule, which may or may not be 
completely base-paired. 
IPS or IPNS - Isopenicillin N synthetase; depending on context, may refer 
to the protein or DNA encoding the protein. 
Isopenicillin N Synthetase - an enzyme, also known as cyclase, which 
catalyzes the formation of isopenicillin N from 
.delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine. 
mRNA - messenger ribonucleic acid. 
Pen DNA - DNA from Penicillium chrysogenum. 
pIPS - isopenicillin N synthetase-encoding DNA of Penicillium chrysogenum. 
pL or .lambda. pL - leftward promoter of bacteriophage lambda. 
Recombinant DNA Cloning Vector - any autonomously replicating or 
integrating agent, including, but not limited to, plasmids, comprising a 
DNA molecule to which one or more additional DNA molecules can be or have 
been added. 
Recombinant DNA Expression Vector - any autonomously replicating or 
integrating agent, including, but not limited to, plasmids, comprising a 
transcription and/or translation activating sequence positioned to drive 
expression of a DNA segment that encodes a polypeptide or RNA of research 
or commercial interest. 
Recombinant DNA Vector - any recombinant DNA cloning or expression vector. 
Restriction Fragment - any linear DNA molecule generated by the action of 
one or more enzymes. 
rRNA - ribosomal ribonucleic acid. 
Sensitive Host Cell - a host cell that cannot grow in the presence of a 
given antibiotic without a DNA segment that confers resistance thereto. 
TcR - the tetracycline resistance-conferring gene. 
Transcription Activating Sequence - a DNA sequence such as a promoter that 
promotes transcription of DNA. 
Transformant - a recipient host cell that has undergone transformation. 
Transformation - the introduction of DNA into a recipient host cell that 
changes the genotype and results in a change in the recipient cell. 
Translation Activating Sequence - a DNA sequence such as a ribosome-binding 
site-encoding sequence that, when translated into mRNA, promotes 
translation of mRNA into protein.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention comprises DNA compounds and recombinant DNA cloning 
and expression vectors that encode the isopenicillin N synthetase activity 
of Aspergillus nidulans. The coding sequence of the isopenicillin N 
synthetase gene of Aspergillus nidulans can be isolated on an -2 kb 
HindIII-BglII restriction fragment of plasmid pOG04. This restriction 
fragment and coding sequence have been further characterized by DNA 
sequencing. The sequence of the A. nidulans isopenicillin N 
synthetase-encoding DNA is depicted below, together with a portion of the 
DNA that flanks the 3' end of the coding region in the A. nidulans genome. 
In the depiction, only the "sense" or coding strand of the double-stranded 
DNA molecule is shown, and the DNA is depicted from left to right in the 
5'.fwdarw.3' orientation. The nucleotide sequence is numbered; the numbers 
appear above the DNA sequence. Immediately below each line of DNA 
sequence, the amino acid residue sequence of the isopenicillin N 
synthetase encoded by the DNA is listed from left to right in the 
amino-terminus.fwdarw.carboxyterminus direction. Each amino acid residue 
appears below the DNA that encodes it. The amino acid residue sequence is 
numbered; the numbers appear below the amino acid residue sequence. 
##STR1## 
wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytidyl, T is 
thymidyl, ALA is an Alanine residue, ARG is an Arginine residue, ASN is an 
Asparagine residue, ASP is an Aspartic Acid residue, CYS is a Cysteine 
residue, GLN is a Glutamine residue, GLU is a Glutamic Acid residue, GLY 
is a Glycine residue, HIS is a Histidine residue, ILE is an Isoleucine 
residue, LEU is a Leucine residue, LYS is a Lysine residue, MET is a 
Methionine residue, PHE is a Phenylaanine residue, PRO is a Proline 
residue, SER is a Serine residue, THR is a Threonine residue, TRP is a 
Tryptophan residue, TYR is a Tyrosine residue, and VAL is a Valine 
residue. 
Those skilled in the art will recognize that the DNA sequence depicted 
above is an important part of the present invention. Due to the degenerate 
nature of the genetic code, which results from there being more than one 
codon for most of the amino acid residues and stop signal, the amino acid 
residue sequence of isopenicillin N synthetase depicted above can be 
encoded by a multitude of different DNA sequences. Because these alternate 
DNA sequences would encode the same amino acid residue sequence of the 
present invention, the present invention further comprises these alternate 
sequences. 
These IPNS-encoding sequences can be conventionally synthesized by the 
modified phosphotriester method using fully protected deoxyribonucleotide 
building blocks. Such synthetic methods are well known in the art and can 
be carried out in substantial accordance with the procedure of Itakura et 
al., 1977, Science 198:1056 and Crea et al., 1978, Proc. Nat. Acad. Sci. 
U.S.A. 75:5765. An especially preferred method of synthesizing DNA is 
disclosed in Hsiung et al., 1983, Nucleic Acid Research 11:3227 and Narang 
et al., 1980, Methods in Enzymology 68:90. In addition to the manual 
procedures referenced above, the DNA sequence can be synthesized using 
automated DNA synthesizers, such as the Systec 1450A or ABS 380A DNA 
Synthesizers. 
In addition to the IPNS-encoding DNA sequences discussed above, there could 
be genetic variants of the isopenicillin N synthetase-encoding DNA of the 
present invention. These genetic variants represent naturally occurring 
genetic diversity and would share substantial DNA and amino acid residue 
sequence homology with the compounds of the present invention and would 
have similar, if not identical, activity, but would differ somewhat in 
nucleotide sequence from the actual compounds of the present invention. 
These genetic variants are equivalent to the compounds of the present 
invention and can be obtained by virtue of homology with the IPNS-encoding 
DNA sequences of the present invention. 
The isopenicillin N synthetase activity-encoding DNA compounds of the 
present invention were isolated from Aspergillus nidulans. A genomic 
library of the total genomic DNA of A. nidulans was constructed, and the 
genomic library was examined for the presence of sequences homologous to 
the Penicillium chrysogenum isopenicillin N synthetase gene encoded on 
plasmid pLC2, a plasmid available from the American Type Culture 
Collection, Rockville, MD 20852, under the accession number ATCC 53334. 
Plasmid pLC2, a restriction site and function map of which is presented in 
FIG. 1 of the accompanying drawings, is disclosed and claimed in U.S. 
patent application Ser. No. 801,523, filed Nov. 25, 1985, attorney docket 
No. X-6932. A variety of the vectors of the genomic library contained DNA 
homologous to the P. chrysogenum isopenicillin N synthetase gene, and DNA 
sequencing revealed that at least one of those vectors encoded the A. 
nidulans isopenicillin N synthetase. The Aspergillus nidulans 
isopenicillin N synthetase gene was then cloned into another vector to 
yield plasmid pOG04, which was transformed into E. coli K12 JM109 host 
cells. The E. coli K12 JM109/pOG04 transformants were deposited and made 
part of the stock culture collection of the Northern Regional Research 
Laboratories (NRRL), Agricultural Research Service, U.S. Department of 
Agriculture, Peoria, Illinois 61604, under the accession number NRRL 
B-18171. A restriction site and function map of plasmid pOG04 is presented 
in FIG. 2 of the accompanying drawings. 
Plasmid pOG04 can be isolated from E. coli K12 JM109/pOG04 by the procedure 
described in Example 1. Useful restriction fragments can be obtained from 
plasmid pOG04 and include an .about.4.5 kb HindIII fragment, which 
comprises the entire IPNS gene, and an .about.1.55 kb ClaI-BglII 
restriction fragment, which encodes all but the amino terminus of IPNS. 
Plasmid pOG04 was used as starting material in the construction of a 
plasmid, designated pOG0216, that drives high-level expression of 
isopenicillin N synthetase in E. coli. Plasmid pOG0216 is constructed by 
inserting the Aspergillus nidulans IPNS coding sequence into a plasmid 
pCZR111-like expression vector that comprises the lamda pL promotor, the 
CI857 temperature sensitive repressor gene, a tetracycline 
resistance-conferring gene, and DNA sequences encoding vector replication 
functions. The use of the type of temperature-inducible expression system 
present on plasmid pCZR111 is described and disclosed in U.S. Patent 
Application Ser. No. 769,221, filed Aug. 26, 1985, attorney docket number 
X-6638, incorporated herein by reference. Essentially, at low temperature 
of about 30.degree. C. the cI857 gene product represses transcription 
driven by the pL promoter, but when the temperature is raised to 
-42.degree. C, the cI857 gene product is inactivated and the pL promoter 
becomes active. Plasmid pCZR111 is available from the NRRL under the 
accession number NRRL B-18249. A restriction site and function map of 
plasmid pCZR111 is presented in FIG. 3 of the accompanying drawings. 
Plasmid pOG0216 comprises the lambda pL promoter and a 2-cistron 
translation activating sequence positioned to drive expression of the 
protein-coding sequence of the Aspergillus nidulans isopenicillin N 
synthetase gene from plasmid pOG04. Two-cistron constructions are 
generally described in Schoner et al., 1984, Proc. Natl. Acad. Sci. 
81:5403-5407 and Schoner et al., 1986, Proc. Natl. Acad. Sci. 
83:8506-8510. The .about.1.6 kb ClaI-BglII restriction fragment of plasmid 
pOG04 comprises all but about the first 36 bp of the protein-coding 
sequence for the isopenicillin N synthetase of Aspergillus nidulans. The 
first 36 bp of the isopenicillin N synthetase coding sequence on plasmid 
pOG0216 is encoded on a synthetic DNA fragment with the following 
sequence: 
##STR2## 
The rest of the isopenicillin N synthetase coding sequence present on 
plasmid pOG0216 was derived directly from plasmid pOG04. A construction 
protocol for plasmid pOG0216 is described in greater detail in Example 2. 
At temperatures of about 42.degree. C., E. coli K12 RV308 (NRRL B-15624) 
cells harboring plasmid pOG0216 express isopenicillin N synthetase at high 
levels, approaching .about.10% of the total cell protein. Crude cell 
extracts from these E. coli K12 RV308/pOG0216 transformants are able to 
catalyze the conversion of 
.delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine into isopenicillin N, 
whereas cell extracts from E. coli K12 RV308 cells cannot catalyze this 
conversion. The method of assay for the conversion reaction is presented 
in Example 3. 
Plasmid pOG0216 provides an efficient means of producing large amounts of 
isopenicillin N synthetase in E. coli. Because E. coli transformants 
containing plasmid pOG0216 express isopenicillin N synthetase at levels 
approaching 10% of total cell protein, and because culturing E. coli is 
less complex than culturing organisms that naturally produce isopenicillin 
N synthetase, E. coli/pOG0216 transformants can be used to produce 
recombinant isopenicillin N synthetase more efficiently and economically 
than non-recombinant or "natural" isopenicillin N synthetase producers. 
The E. coli K12/pOG0216 transformants of the present invention, by 
producing such high levels of isopenicillin N synthetase, allow for the 
isolation of the isopenicillin N synthetase encoded on the Aspergillus 
nidulans genome in substantially pure form. 
Isopenicillin N synthetase can be used to produce isopenicillin N from 
6-(L-o-aminoadipyl)-L-cysteinyl-D-valine in a cell-free system, as 
described in Example 3. Isopenicillin N is not only a useful antibiotic, 
but also is the starting material for the production of such important 
antibiotics as penicillin N, cephalexin, and other cephalo-sporins as 
described in U.S. Pat. No. 4,307,192. Another important use of 
isopenicillin N synthetase is for condensing tripeptides other than 
.delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine into novel 
.beta.-lactam derivatives. 
Cell-free extracts of penicillin-producing organisms can be used to 
synthesize unnatural (not produced in nature) .beta.-lactams. The E. coli 
expression vectors of the present invention provide an inexpensive and 
efficient method of obtaining isopenicillin N synthetase, both in crude 
cell extracts and in substantially purified form, which can be used in 
vitro to condense tripeptides that do not naturally occur in nature to 
form novel antibiotics or antibiotic core structures. 
Plasmid pOG0216 is especially preferred for driving expression of IPNS in 
E. coli not only because of the high expression levels achieved when using 
the plasmid but also because of the selectable marker present on the 
plasmid. Many recombinant DNA vectors encode a .beta.-lactamase, so that 
cells transformed with such vectors can grow in the presence of certain 
.beta.-lactam antibiotics, such as ampicillin. However, if one desires to 
use a cell-free extract containing IPNS for purposes of constructing 
.beta.-lactams, one would not want the extract to contain .beta.-lactamase 
activity. Thus, plasmid pOG0216 does not encode a .beta.-lactamase for a 
selectable marker but rather encodes the tetracycline 
resistance-conferring gene, the gene product of which is non-reactive with 
.beta.-lactams. 
The IPNS expression vectors of the present invention are not limited to a 
particular selectable marker. Those skilled in the art recognize that many 
selectable markers are suitable for use on IPNS expression vectors. Such 
selectable markers include genes that confer kanamycin resistance, i.e., a 
selectable marker on TN903, and genes that confer chloramphenicol 
resistance, i.e., a selectable marker on plasmids pACYC184 and pBR325. 
The vectors of the present invention include vectors that drive expression 
of IPNS in .beta.-lactam producing organisms. The .beta.-lactamase gene 
cannot be used as a selectable marker in a .beta.-lactam-producing 
microorganism. The .beta.-lactamase gene can be present on vectors of the 
present invention designed for use in .beta.-lactam-producing organisms 
simply because of its utility as a selectable marker in E. coli. Many 
vectors designed for .beta.-lactam-producing organisms also are 
constructed so as to replicate in E. coli for ease of plasmid preparation. 
Certain .beta.-lactam-producing organisms, such as Cephalosporium 
acremonium, are eukaryotic cells, but nevertheless, the prokaryotic 
.beta.-lactamase gene derived from plasmid pBR322 seems to function in 
some eukaryotic host cells. See Marczynski and Jaehning, 1985, Nuc. Acids 
Res. 13(23):8487-8506 and Breunig et al., 1982, Gene 20:1-10. To avoid the 
possibility of introducing a .beta.-lactamase gene that could possibly 
express in an organism transformed to obtain greater 
.beta.-lactam-producing ability, the present invention also comprises 
vectors that utilize a selectable marker other than the .beta.-lactamase 
gene, such as a chloramphenicol acetyltransferase-encoding gene. 
As stated above, a .beta.-lactamase gene cannot be used as a selectable 
marker in Cephalosporium acremonium, Penicillium chrysogenum, Streptomyces 
clavuligerus, or Aspergillus nidulans, nor do these organisms encode an 
endogenous .beta.-lactamase. However, many E. coli strains, even those 
sensitive to .beta.-lactams, do encode and express, at low levels, an 
endogenous .beta.-lactamase, i.e., the E. coli ampC gene product. See 
Juarin et al., 1981, Proc. Natl. Acad. Sci. 78(8):4897-4901 and Grundstrom 
et al., 1982, Proc. Natl. Acad. Sci. 79:1111-1115. The presence of the 
ampC gene product in crude cell extracts of recombinant E. coli cells 
containing an IPNS expression vector could lead to degradation of 
.beta.-lactams prepared using that extract. To avoid such degradation, E. 
coli K12 RV308 was subjected to mutagenesis to obtain a strain, designated 
E. coli K12 A85892, that does not express a .beta.-lactamase activity 
(unless the activity is encoded on a recombinant vector present in the 
cell). E. coli K 12 A85892 can be obtained from the Northern Regional 
Research Center under the accession number NRRL B-18096. 
The search for unnatural tripeptides that will serve as substrates for 
isopenicillin N synthetase can be complemented by a search for mutant 
isopenicillin N synthetases that will accept unnatural tripeptides as 
substrate. The present invention provides the starting material for such a 
search for a mutant isopenicillin N synthetase. E. coli is the best host 
for mutational cloning experiments, and the E. coli expression vectors of 
the present invention can be readily mutated by procedures well known in 
the art, such as, for example, treatment with radiation (X-ray or UV) or 
chemical mutagens (such as ethylmethanesulfonate, nitrosoguanidine, or 
methyl methanesulfonate) or site-specific mutagenesis, to obtain mutant 
enzymes that recognize unnatural tripeptides as substrate and catalyze the 
condensation of those unnatural tripeptides to unnatural .beta.-lactams. 
The present invention is not limited to the particular vectors exemplified 
herein. The DNA compounds of the present invention encode the 
isopenicillin N synthetase activity of Aspergillus nidulans and can be 
used to isolate homologous DNA compounds from other Aspergillus strains 
that encode genetic variants of the isopenicillin N synthetase of the 
present invention. Consequently, the present invention comprises DNA 
compounds homologous to the isopenicillin N synthetase-encoding DNA on 
plasmids pOG04 and pOG0216 that encode isopenicillin N synthetase 
activity. The DNA compounds of the present invention can be used to 
construct expression vectors that drive expression of isopenicillin N 
synthetase in any host cell in which the expression vector replicates or 
integrates and in which the transcription and translation activating 
sequence used to express the isopenicillin N synthetase activity 
functions. 
The E. coli expression vectors of the invention are not limited to the 
specific vectors exemplified herein. The present invention comprises any 
E. coli expression plasmid or vector that drives expression of 
isopenicillin N synthetase in E. coli. Thus, the present invention 
comprises expression vectors that drive expression of isopenicillin N 
synthetase and utilize a replicon functional in E. coli, such as, for 
example, a runaway replicon or a replicon from such plasmids as pBR322, 
pACYC184, F, ColV-K94, R1, R6-5, or R100. Nor is the present invention 
solely limited to plasmid vectors, for the present invention also 
comprises expression vectors that express isopenicillin N synthetase 
activity and utilize integration or viral replication to provide for 
replication and maintenance in the host cell. 
The present invention is not limited to a particular transcription and 
translation activating sequence to drive expression of the isopenicillin N 
synthetase activity-encoding DNA. The present invention comprises the use 
of any transcription and translation activating sequence to express 
isopenicillin N synthetase in E. coli. Many transcription and translation 
activating sequences that function in E. coli are known and are suitable 
for driving expression of isopenicillin N synthetase activity in E. coli. 
Such transcription and translation activating sequences include, but are 
not limited to, the lpp, lac, trp, tac, .lambda.p.sub.L, and 
.lambda.p.sub.R transcription and translation activating sequences. 
In addition to the various E. coli replicons and transcription and 
translation activating sequences exemplified above, replicons and 
transcription and translation activating sequences from other organisms 
can be ligated to the present isopenicillin N synthetaseencoding DNA 
compounds to form expression vectors that drive expression of 
isopenicillin N synthetase activity in host cells in which the replicon 
and activating sequence function. Although E. coli is the host best suited 
for isopenicillin N synthetase production and subsequent purification for 
in vitro use, vectors that drive expression of isopenicillin N synthetase 
activity in host cells other than E. coli are also useful, especially for 
purposes of increasing the .beta.-lactam antibiotic-producing ability and 
efficiency of a given organism. 
A variety of organisms produce -lactam antibiotics. The following Table 
presents a non-comprehensive list of .beta.-lactam antibiotic-producing 
organisms. 
TABLE I 
______________________________________ 
.beta.-Lactam Antibiotic-Producing Organisms 
Organism Antibiotic 
______________________________________ 
Agrobacterium various .beta.-lactams 
Aspergillus nidulans 
various .beta.-lactams 
Cephalosporium 
acremonium penicillins and 
cephalosporins 
Chromobacterium various .beta.-lactams 
Gluconobacter various .beta.-lactams 
Nocardia 
lactamdurans cephamycin C 
uniformis nocardicin 
Penicillium 
chrysogenum various penicillins and 
other .beta.-lactams 
Serratia various .beta.-lactams 
Streptomyces 
antibioticus clavulanic acid 
argenteolus asparenomycin A, 
MM 4550, and MM 13902 
cattleya thienamycin 
chartreusis SF 1623 and 
cephamycin A and B 
clavuligerus PA-32413-I, cephamycin C, 
A16886A, penicillins, 
cephalosporins, 
clavulanic acid, 
and other clavams 
fimbriatus cephamycin A and B 
flavovirens MM 4550 and MM 13902 
flavus MM 4550 and MM 13902 
fulvoviridis MM 4550 and MM 13902 
griseus cephamycin A and B 
and carpetimycin A and B 
halstedi cephamycin A and B 
heteromorphus C2081X and 
cephamycin A and B 
hygroscopicus deacetoxycephalosporin C 
lipmanii cephamycin, penicillin N, 
7-methoxycephalosporin C, 
A16884, MM4550, MM13902 
olivaceus epithienamycin F, 
MM 4550, and MM 13902 
panayensis C2081X and 
cephamycin A and B 
pluracidomyceticus 
pluracidomycin A 
rochei cephamycin A and B 
sioyaensis MM 4550 and MM 13902 
sp. OA-6129 OA-6129A 
sp. KC-6643 carpetimycin A 
tokunomensis asparenomycin A 
viridochromogenes 
cephamycin A and B 
wadayamensis WS-3442-D 
______________________________________ 
Many of the foregoing .beta.-lactam antibiotic-producing organisms are used 
in the pharmaceutical industry for purposes of antibiotic production. The 
antibiotic-producing ability of these organisms can be increased and made 
more efficient by increasing the intracellular concentration of 
rate-limiting antibiotic biosynthetic enzymes during the fermentation. The 
isopenicillin N synthetase activity-encoding DNA compounds of the present 
invention can be used to construct expression vectors. When these IPNS 
expression vectors are transformed into a host cell that produces a 
.beta.-lactam antibiotic via an intermediate reaction involving 
isopenicillin N synthetase activity, the intracellular concentration of 
isopenicillin N synthetase activity is increased. Provided that IPNS 
activity is the rate-limiting factor of the .beta.-lactam biosynthesis in 
the untransformed host cell, host cells containing these IPNS expression 
vectors produce more .beta.-lactam antibiotic than their untransformed 
counterparts. 
A vector that will increase the intracellular concentration of 
isopenicillin N synthetase activity of a given host cell into which the 
vector is transformed requires the following elements: (1) an 
isopenicillin N synthetase activity-encoding DNA compound; (2) a 
transcription and translation activating sequences that not only functions 
in the host cell to be transformed, but also is positioned in the correct 
orientation and position to drive expression of the isopenicillin N 
synthetase activity-encoding DNA; and (3) replication or integration 
functions that provide for maintenance of the vector in the host cell. The 
frequency of integration of a DNA vector often is quite dependent on 
activities encoded by the host cell; however, it is often observed that 
certain DNA sequences (i.e., sequences from viruses and phages that 
facilitate integration and sequences homologous to the host's genomic 
DNA), when present on a recombinant DNA vector facilitate integration. Of 
course, an IPNS expression vector could also comprise an antibiotic 
resistance-conferring gene or some other element that provides a means of 
selecting for host cells which contain the vector, but such selectable 
elements may be neither necessary nor desired when the vector integrates 
into the chromosomal DNA of the host cell. 
A variety of the plasmids of the present invention are useful for 
increasing the intracellular concentration of isopenicillin N synthetase 
activity in a .beta.-lactam antibiotic-producing cell. Plasmid pOG04 
comprises the intact isopenicillin N synthetase gene of Aspergillus 
nidulans, so transformation of A. nidulans via chromosomal integration of 
plasmid pOG04 leads to increased copy number of the isopenicillin N 
synthetase gene and thus leads to increased intracellular concentration of 
the enzyme. European Patent Publication number 0191221A1, incorporated 
herein by reference, describes various transformation protocols that can 
be used to transform Aspergillus with a recombinant DNA vector. The 
Aspergillus nidulans isopenicillin N synthetase gene also is believed to 
function in Cephalosporium acremonium and Penicillium chrysogenum. 
Consequently, transformation of P. chrysogenum or C. acremonium via 
chromosomal integration of plasmid pOG04 leads to increased copy number of 
the isopenicillin N synthetase gene and thus leads to increased 
intracellular concentration of the enzyme. 
However, the Aspergillus nidulans IPNS coding sequence of the invention can 
also be put under the control of transcription and translation activating 
sequences derived from Penicillium and Cephalosporium to construct a 
recombinant gene especially for use in these organisms. U.S. patent 
application Ser. No. 06/895,008, filed Aug. 8, 1986, attorney docket No. 
X-6722B, incorporated herein by reference, discloses the transcription and 
translation activating sequences of the C. acremonium IPNS gene, which can 
be fused to the A. nidulans IPNS coding sequence of the present invention 
to create a recombinant IPNS gene that drives expression (when 
incorporated into an expression vector and the vector introduced into 
Cephalosporium) of the A. nidulans IPNS in Cephalosporium. Likewise, U.S. 
patent application Ser. No. 06/801,523, filed Nov. 25, 1985, incorporated 
herein by reference, discloses the transcription and translation 
activating sequences of the P. chrysogenum IPNS gene, which can be used as 
described above to construct Penicillium vectors that drive expression of 
A. nidulans IPNS. 
The present invention results from the cloning of an intact, functional, 
Asperqillus nidulans DNA sequence that encodes not only the amino acid 
sequence of isopenicillin N synthetase but also the transcription and 
translation activating sequence necessary to drive expression of 
isopenicillin N synthetase in A. nidulans . Likewise, the isopenicillin N 
synthetase gene of the present invention comprises the sequences located 
downstream of the coding region that are responsible for terminating 
transcription and for providing the mRNA polyadenylation and processing 
signals. These 5' and 3' regulatory elements comprise an important aspect 
of the present invention. 
Because plasmid pOG04 comprises .about.0.45 kb of the genomic DNA that was 
located upstream of the isopenicillin N synthetase-encoding DNA in the 
Aspergillus nidulans genome, plasmid pOG04 necessarily comprises the 
transcription and translation activating sequence of the A. nidulans 
isopenicillin N synthetase gene. Most transcription and translation 
activating sequences are encoded upstream of the DNA to be activated, 
although some ribosomal RNA-encoding DNA sequences are activated by 
transcription activating sequences that are not located upstream of the 
coding region. "Upstream," in the present context, refers to DNA in the 5' 
direction from the 5' end of the coding strand of the isopenicillin N 
synthetase-encoding DNA. 
The Aspergillus nidulans transcription and translation activating sequence 
encoded on plasmid pOG04 is correctly positioned to drive expression of 
the isopenicillin N synthetase activity-encoding DNA. In the construction 
of plasmid pOG04, no deletions or insertions affecting the transcription 
and translation activating sequence were introduced in the DNA flanking 
the 5' end of the coding strand of the isopenicillin N synthetase 
activity-encoding DNA. Because the Aspergillus nidulans transcription and 
translation activating sequence located on plasmid pOG04 can be used to 
drive expression of a wide variety of DNA sequences, the activating 
sequence comprises an important part of the present invention. The 
activating sequence of the A. nidulans isopenicillin N synthetase gene can 
be isolated on the .about.450 bp HindIII-ClaI restriction fragment located 
immediately upstream of and adjacent to the isopenicillin N synthetase 
activity-encoding DNA on plasmid pOG04. The ClaI site encodes amino acid 
residues 12 and 13 of the isopenicillin N synthetase protein, so the amino 
terminal coding region of the isopenicillin N synthetase is also contained 
on this HindIII-ClaI fragment. Any restriction fragment that comprises the 
aforementioned .about.450 bp HindIII-ClaI restriction fragment necessarily 
comprises the A. nidulans transcription and translation activating 
sequence of the present invention. 
The DNA sequence of the Aspergillus nidulans transcription and translation 
activating sequence encoded on plasmid pOG04 is presented below. This 
sequence can be chemically synthesized or isolated from plasmid pOG04. To 
clarify how the activating sequence is oriented in plasmid pOG04, the 
restriction fragment is illustrated with a single-stranded DNA overlap 
characteristic of restriction enzyme HindIII and the translation 
initiation codon is included. 
##STR3## 
The Aspergillus nidulans transcription and translation activating sequence 
can be used to drive expression of any DNA sequence in A. nidulans and 
other Aspergillus species. The transcriptional promoter from the 
Aspergillus nidulans isopenicillin N synthetase gene can be fused to 
protein coding regions in several useful ways. For example, the majority 
of the 5' noncoding information can be isolated on an .about.400 bp 
HindIII-BamHI fragment from pOG04. The remaining .about.55 bp of 5' 
noncoding information before the 5'-ATG translation initiation codon can 
be appended through use of a synthetic linker, essentially as described in 
Example 2. The synthetic linker can incorporate useful restriction sites 
at the translation initiation site, such as NdeI or NcoI, to facilitate 
fusion of the A. nidulans promoter to the protein coding region of 
interest. The protein coding region can be similarly adapted to contain 
compatible ends for convenient ligation using strategies which will be 
different for different protein coding regions, but an exemplary strategy 
is outlined in Example 2, which describes the protocol for fusing the A. 
nidulans isopenicillin N synthetase protein-coding region to the E. coli 
transcriptional promoter and translation activating sequence on plasmid 
pCZR111. 
Similar strategies allow the A. nidulans isopenicillin N synthetase protein 
coding region to be joined to transcriptional promoters and translation 
activating sequences from any organism of interest. For example, a 
transcriptional promoter from the organism of interest can be 
reconstructed so that the 5'-ATG translation initiation site is nested 
within an NcoI restriction site, 5 -CCATGG-3'. An A. nidulans 
isopenicillin N synthetase protein coding region containing an NcoI 
restriction site at the translation initiation site, as taught in Example 
2, can then be simply joined to the promoter from the organism of interest 
to produce a hybrid gene that will drive expression of the A. nidulans 
isopenicillin N synthetase in any organism in which the activating 
sequence functions. The flanking ends of the hybrid gene can be similarly 
adapted to allow insertion into a vector suitable for the organism of 
interest. 
Plasmid pOG04 also comprises the 3' regulatory sequences of the Aspergillus 
nidulans isopenicillin N synthetase gene. Usually, the sequences 
responsible for transcription termination, mRNA polyadenylation, and mRNA 
processing are encoded within the region .about.500 bp downstream of the 
stop codon of the coding region of a gene. Therefore, the .about.0.65 kb 
HincII-BamHI restriction fragment that comprises the isopenicillin N 
synthetase carboxy-terminal-encoding DNA and downstream sequences also 
comprises the transcription termination and mRNA polyadenylation and 
processing signals of the A. nidulans isopenicillin N synthetase gene. 
The transcription termination sequence in plasmid pOG04 can be appended to 
other recombinant gene constructions to facilitate transcription 
termination in those gene constructs. For example, a HincII restriction 
site (located at about the codons for amino acid residues 286 and 287 of 
the A. nidulans isopenicillin N synthetase) is convenient for isolating 
the transcription termination sequences, both because the HincII cleavage 
site is near the translation termination site (translation terminates 
after amino acid residue 331) and because HincII cleavage produces flush 
ends that facilitate linkage to various gene constructs. A BamHI 
restriction enzyme recognition site located about 510 bp downstream of the 
translation termination site provides a convenient distal or downstream 
end, allowing the transcription terminator to be isolated on an .about.650 
bp HincII-BamHI restriction fragment from plasmid pOG04. 
Expression of a given DNA sequence on a recombinant DNA expression vector 
can be enhanced by placing a transcription termination and mRNA 
polyadenylation and processing signal at the 3' end of the coding strand 
of the coding region to be expressed. The present invention provides a 
transcription termination and mRNA polyadenylation and processing signal 
that can be used for the purposes of increasing expression of any gene 
product from a recombinant DNA vector in Aspergillus and related host 
cells. 
The present invention provides the coding sequence for the isopenicillin N 
synthetase gene of Aspergillus nidulans and provides a number of 
expression vectors that drive expression of that gene in host cells such 
as E. coli. Production of isopenicillin N synthetase in E. coli allows for 
high-level expression and easy isolation of the enzyme so that the enzyme 
can be used to catalyze the condensation of novel tripeptides into novel 
antibiotic core structures in vitro. Transformation of A. nidulans, 
Cephalosporium acremonium, Penicillium chrysogenum, and other 
.beta.-lactam antibiotic-producing host cells with expression vectors of 
the present invention that drive expression of isopenicillin N synthetase 
in the host cell of interest leads to higher levels of isopenicillin N 
synthetase and thus leads to higher levels of antibiotic in the 
transformed cell. 
The following Examples are provided to further illustrate and exemplify the 
present invention but are in no way intended to limit the scope of the 
present invention. 
EXAMPLE 1 
Culture of E. coli K12 JM109/pOG04 and Isolation of Plasmid pOG04 
A. Culture of E. coli K12 JM109/pOG04 
A lyophil of E. coli K12 JM109/pOG04 is obtained from the Northern Regional 
Research Laboratories, Peoria, Ill. 61604, under the accession number NRRL 
B-18171. The lyophil can be directly used as the "culture" in the process 
described below. 
One liter of L-broth (10 g tryptone, 10 g NaCl, and 5 g yeast extract per 
liter) containing 50 .mu.g/ml ampicillin was inoculated with a culture of 
E. coli K12 JM109/pOG04 and incubated with aeration at 37.degree. C. until 
the optical density at 590 nm (O.D..sub.590) was .about.1 absorbance unit, 
at which time 150 mg of chloramphenicol were added to the culture. The 
incubation was continued for about 16 hours; the chloramphenicol addition 
inhibits protein synthesis, and thus inhibits further cell division, but 
allows plasmid replication to continue. 
B. Isolation of Plasmid pOG04 
The culture prepared in Example 1A was centrifuged in a Sorvall GSA rotor 
(DuPont Co., Instrument Products, Biomedical Division, Newtown, Conn. 
06470) at 6000 rpm for 5 minutes at 4.degree. C. The resulting supernatant 
was discarded, and the cell pellet was washed in 40 ml of TES buffer (10 
mM Tris-HCl, pH=7.5; 10 mM NaCl; and 1 mM EDTA) and then repelleted. The 
supernatant was again discarded, and the cell pellet was frozen in a dry 
ice-ethanol bath and then thawed. The thawed cell pellet was resuspended 
in 10 ml of a solution of 25% sucrose and 50 mM EDTA. About 1 ml of a 5 
mg/ml lysozyme solution; 3 ml of 0.25 M EDTA, pH=8.0; and 100 .mu.l of 10 
mg/ml RNAse A, were added to and mixed with the solution, which was then 
incubated on ice for 15 minutes. Three ml of lysing solution (prepared by 
mixing 3 ml of 10% Triton-X 100; 75 ml of 0.25 M EDTA, pH=8.0; 15 ml of 1 
M Tris-HCl, pH=8.0; and 7 ml of water) were added to the lysozyme-treated 
cells, mixed, and the resulting solution incubated on ice for another 15 
minutes. The lysed cells were frozen in a dry ice-ethanol bath and then 
thawed. 
The cellular debris was removed from the solution by centrifugation at 
25,000 rpm for 40 minutes in an SW27 rotor (Beckman, 7360 N. Lincoln Ave., 
Lincolnwood, Ill. 60646) and by extraction with buffered phenol. About 
30.44 g of CsCl and .about.1 ml of a 5 mg/ml ethidium bromide solution 
were added to the solution; then, the volume was adjusted to 40 ml and 
decanted into a VTi50 ultra-centrifuge tube (Beckman). The tube was 
sealed, and the solution was centrifuged in a VTi50 rotor at 42,000 rpm 
for .about.16 hours. The resulting plasmid band, visualized with 
ultraviolet light, was isolated and then placed in a Ti75 tube (volume 
adjustments were made using TES containing 0.761 g/ml CsCl) and rotor 
(Beckman) and centrifuged at 50,000 rpm for 16 hours. The plasmid band was 
again isolated, extracted with salt-saturated isopropanol to remove the 
ethidium bromide, and diluted 1:3 with TES buffer. Two volumes of ethanol 
were then added to the solution, which was then incubated overnight at 
-20.degree. C. The plasmid DNA was pelleted by centrifuging the solution 
in an SS34 rotor (Sorvall, DuPont Co., Newton, Conn. 06470) for 15 minutes 
at 10,000 rpm. 
The .about.1 mg of plasmid pOG04 DNA obtained by this procedure was 
suspended in 1 ml of TE buffer (10 mM Tris-HCl, pH=8.0, and 1 mM EDTA) and 
stored at -20.degree. C. A restriction site and function map of plasmid 
pOG04 is presented in FIG. 2 of the accompanying drawings. 
EXAMPLE 2 
Construction of Plasmid pOG0216 
Plasmid pOG0216 was constructed by ligating together the following four DNA 
fragments: an .about.1.6 kb ClaI-BglII restriction fragment from plasmid 
pOG04 DNA that contains all but the IPNS amino-terminus-encoding portion 
of the Aspergillus nidulans IPNS coding sequence; an NcoI-ClaI 
double-stranded DNA linker that reconstructs the coding sequence for the 
amino terminus of Aspergillus nidulans IPNS; an .about.5.3 kb EcoRI-BamHI 
restriction fragment from plasmid DNA of a pCZR111-derivative that 
contains the DNA sequences coding for the cI857 repressor, the plasmid 
origin of replication, and the tetracycline resistance-conferring gene; 
and an .about.500 bp EcoRI-NcoI restriction fragment from plasmid DNA of 
the aforementioned pCZR111 derivative expression vector. This pCZR111 
derivative differs from the parent plasmid pCZR111 only in the translation 
activating sequence and downstream protein-coding sequence. One skilled in 
the art will recognize that different plasmids can share common DNA 
sequences encoding promoters, translation activating sequences, origins of 
replication, and antibiotic resistance-conferring genes. It is often 
matters of convenience rather than design that dictate choices for sources 
of DNA sequences, such as which plasmids are prepared and ready for use. 
To facilitate description, construction of plasmid pOG0216 is described 
using plasmid pCZR111 DNA as the source for the DNA sequences encoding the 
.lambda.pL promoter, the plasmid origin of replication, the cI857 gene, 
and the tetracycline resistance-conferring gene; plasmid pOG04 as the 
source plasmid for all but the amino terminus-encoding portion of 
Aspergillus nidulans IPNS coding sequence; and a double-stranded synthetic 
DNA linker as the source of DNA coding for the two cistron translation 
activating sequence as well as the amino terminus-encoding portion of the 
Aspergillus nidulans IPNS coding sequence. 
A. Culture of E. coli K12 RV308/pCZR111 and Isolation of Plasmid pCZR111 
A lyophil of E. coli K12 RV308/pCZR111 is obtained from the NRRL under the 
accession number NRRL B-18249. The lyophil is reconstituted in L broth; 
the resulting culture is used to prepare plasmid pCZR111 DNA in 
substantial accordance with the procedure described in Example 1. However, 
because the selectable marker on plasmid pCZR111 is the tetracycline 
resistance-conferring gene, the culture medium contains 10 .mu.g/ml 
tetracycline and no ampicillin. In addition, the culture is incubated at 
25.degree.-30.degree. C., instead of 37.degree. C., to prevent 
transcription from the lambda pL promoter. About 1 mg of plasmid pCZR111 
DNA is obtained by this procedure and dissolved in .about.1 ml of 0.1 X TE 
buffer. A restriction site and function map of plasmid pCZR111 is 
presented in FIG. 4 of the accompanying drawings. B. Isolation of the 
.about.5.8 kb XbaI-BamHI Restriction Fragment of Plasmid pCZR111 
Approximately 25 .mu.g (in 25 .about.1 of 0.1 X TE buffer) of the plasmid 
pCZR111 DNA prepared in Example 2A are added to and mixed with 40 .about.1 
of 10X XbaI buffer (500 mM Tris-HCl, pH =8.0; 500 mM NaCl; and 100 mM 
MgCl.sub.2), 335 .about.l of water, 2 .about.l (50 units) of restriction 
enzyme BamHI, and 3 .about.l (60 units) of restriction enzyme XbaI. Unless 
otherwise noted, restriction enzymes referred to herein can be obtained 
from New England Biolabs, 32 Tozer Road, Beverly, Mass. 01915. Unit 
definitions herein correspond to the particular manufacturer's unit 
definitions. The resulting reaction is incubated at 37.degree. C. for 90 
minutes. The DNA is concentrated by precipitation with ethanol and NaOAc 
and collected by centrifugation. The DNA pellet is resuspended in 
.about.80 .mu.l of water and .about.20 .about.l of 5X loading buffer (70% 
glycerol, 50 mM EDTA, and 1 mg/mL bromphenol blue) and electrophoresed on 
an .about.1.0% agarose gel until the desired .about.5.8 kb XbaI-BamHI 
restriction fragment is clearly separated from the other digestion 
product, an .about.600 bp XbaI-BamHI restriction fragment. Visualization 
of the electrophoresed DNA is accomplished by staining the gel in a dilute 
solution of ethidium bromide .about.1 .mu.g/.mu.l) and exposing the gel to 
long-wave UV light. 
The desired fragment is located, excised from the gel, and the .about.5.8 
kb XbaI-BamHI restriction fragment recovered using a D-Gel=DNA 
electroeluter (Epigene, Box 4817, Baltimore, MD, 21211) in substantial 
accordance with the manufacturer's directions. Two volumes of ethanol are 
added to the DNA, which is recovered from the eluter in 500 .mu.l of 1 M 
NaCl. The resulting solution is incubated at -20.degree. C. for .about.5 
hours, then centrifuged at 15,000 rpm for 20 minutes. The DNA pellet is 
rinsed first with 70% ethanol and then with 100% ethanol, dried, and 
resuspended in 20 .mu.l of 0.1 X TE buffer. This solution constitutes 
about 2 .mu.g of the desired .about.5.8 kb XbaI-BamHI restriction fragment 
and is stored at -20.degree. C. 
C. Preparation of Plasmid pOG04 DNA from E. coli K12 GM48 
The ClaI restriction site located at about the codons for amino acid 
residues 13 and 14 of the Aspergillus nidulans isopenicillin N synthetase 
coding sequence is methylated in dam.sup.+ E coli strains, because the "G" 
residue preceding the ClaI recognition sequence produces the 5'-GATC-3' 
sequence that is methylated at the "A" residue by the dam gene product. 
Because ClaI restriction enzyme will not cleave when either "A" residue in 
the recognition sequence is methylated, it was necessary to prepare 
plasmid pOG04 DNA from a dam mutant strain of E. coli, such as E. coli K12 
GM48, to isolate the .about.1.6 kb ClaI-BglII restriction fragment of 
plasmid pOG04 for use in the construction of plasmid pOG0216. 
E. coli K12 GM48 is obtained from the Northern Regional Research 
Laboratories under accession number NRRL B-15725. The lyophil of E. coli 
K12 GM48 cells is used to inoculate .about.100 ml of L broth, and the 
culture is incubated at 37.degree. C. until the absorbance at 600 nm is 
about 0.5-0.7. A variety of procedures exist for preparing E. coli cells 
competent for transformation. One such procedure is described below. The 
cells are centrifuged at 5,000Xg for 5 minutes, washed in 50 ml of sterile 
cold 10 mM NaCl, and centrifuged again. The cells are then gently 
resuspended in 2-5 ml of sterile, cold 75 mM CaClz and are then competent 
for transformation. About .about.100 mg of the plasmid pOG04 DNA prepared 
in Example 1A were added to 200 .about.1 of the E. coli GM48 cells, and 
the mixture was incubated on ice for 1 hour. The mixture was then 
incubated at 42.degree. C. for 2 minutes, then diluted into 2 ml of L 
broth and incubated for 1 hour at 37.degree. C. without shaking. Aliquots 
were plated on L agar plates containing 100 .mu.g/ml ampicillin, and the 
plates were incubated at 37.degree. C. overnight. Individual 
ampicillin-resistant colonies were grown, and plasmid pOG04 DNA was 
prepared from the E. coli GM48/pOG04 transformants in substantial 
accordance with the procedure described in Example 1. 
D. Isolation of the .about.1.6 kb ClaI-BglII Restriction Fragment of 
Plasmid pOG04 that Encodes Isopenicillin N Synthetase 
Approximately 25 .mu.g (in 25 .about.1 of TE buffer) of the plasmid pOG04 
DNA prepared in Example 2C were dissolved in 10 .mu.l of 10X ClaI buffer 
(0.5 M NaCl; 0.06 M Tris-HCl, pH=7.9; 0.06 M MgClz; 1 mg/ml BSA) and 60 
.mu.l of H.sub.2 O. About 5 .mu.l (50 units) of restriction enzyme ClaI 
were added to the solution of plasmid pOG04 DNA, and the resulting 
reaction was incubated at 37.degree. C. for two hours. Then, about 5 .mu.l 
(50 units) of restriction enzyme BglII were added to the reaction mixture 
and the resulting reaction was incubated at 37.degree. C. for an 
additional 2 hours. The ClaI-BglII-digested DNA obtained was loaded onto a 
1% agarose gel, and the desired .about.1.6 kb ClaI-BglII restriction 
fragment was isolated, purified, and prepared for ligation. Approximately 
2 .mu.g of the desired fragment was obtained, suspended in 20 .about.1 of 
TE buffer, and stored at -20.degree. C. 
E. Construction of an XbaI-ClaI Linker 
Two complementary single-stranded DNA fragments can be synthesized on an 
automated DNA synthesizer and subsequently annealed to form a 
double-stranded fragment. Many DNA synthesizing instruments are known in 
the art and are suitable for making the fragments. One such instrument is 
an ABS 380A DNA Synthesizer (Applied Biosystems, Inc. 850 Lincoln Centre 
Dr., Foster City, CA 94404). In addition, the fragment can also be 
conventionally prepared in substantial accordance with the procedures of 
Itauura et al., 1977, Science, 198:1056 and Crea et al., 1978, Proc. Natl. 
Acad. Sci. U.S.A. 75:5765. 
The following linkers are synthesized: 
##STR4## 
About 100 pmols of each single-stranded DNA fragment are kinased in 
separate 50 .mu.l reactions containing the single-stranded DNA fragment, 5 
.mu.l of 10X ligase buffer, 5 .mu.l of 5 mM ATP, water to adjust the final 
reaction volume to 50 .mu.l and 1 .mu.l (.about.10 Richardson units) of T4 
polynucleotide kinase. The reactions are incubated at 37.degree. C. for 30 
minutes. The two reactions are then combined, and the resulting 100 .mu.l 
of reaction mixture are heated to 90.degree. C. and allowed to cool slowly 
to 4.degree. C. to optimize annealing of the complementary single-stranded 
fragments. The resulting double-stranded DNA fragment, the linker, is: 
##STR5## 
This linker joins the XbaI end of the pCZR111-derived fragment to the ClaI 
end of the plasmid pOG04-derived fragment, reconstructs the coding 
sequence for the amino terminus of Aspergillus nidulans isopenicillin N 
synthetase, and also encodes the translation activating DNA sequence. 
F. Final Construction of Plasmid pOG0216 
One .mu.l of the .about.5.8 kb XbaI-BamHI restriction fragment of plasmid 
pCZR111, 1 .mu.l of the .about.1.6 kb ClaIBglII restriction fragment of 
plasmid pOG04, and .about.1 .mu.l (.mu.l pmole) of the annealed synthetic 
linker are ligated to form plasmid pOG0216. The total reaction volume is 
20 .mu.l and contains the DNA fragments, 2 .mu.l of 10 X ligase buffer 
(0.5 M Tris-HCl, pH =7.5, 100 .mu.M MgCl.sub.2), 2 .mu.l of 5 mM ATP, 1 
.mu.l of a 6 .mu.g/.mu.l BSA solution, 9 .mu.l of water, and 1 .mu.l (1 
Weiss unit) of T4 DNA ligase (Boehringer-Mannheim Biochemicals (BMB), P.O. 
Box 50816, Indianapolis, Ind. 46250). The reaction is incubated .about.18 
hours at 15.degree. C. The ligated DNA constitutes the desired plasmid 
pOG0216. A restriction site and function map of plasmid pOGO216 is 
presented in FIG. 3 of the accompanying drawings. 
EXAMPLE 3 
Construction of E. coli K12 RV308/pOGO216 and Assay of E. coli-Produced 
Isopenicillin N Synthetase 
A. Construction of E. coli K12 RV308/pOGO216 
E. coli K12 RV308 cells competent for transformation can be prepared as 
follows. A 50 ml culture of E. coli K12 RV308 (NRRL B-15624) in L-broth is 
grown to an O.D..sub.590 of .about.0.5 absorbance units. The culture is 
chilled on ice for ten minutes, and the cells are collected by 
centrifugation. The cell pellet is resuspended in 25 ml of cold 100 mM 
CaCl.sub.2 and incubated on ice for 25 minutes. The cells are once again 
pelleted by centrifugation, and the pellet is resuspended in 2.5 ml of 
cold 100 mM CaCl.sub.2 and incubated on ice overnight. 
Two hundred .mu.l of this cell suspension are mixed with the ligated DNA 
prepared in Example 2F and then incubated on ice for 20 minutes. The cells 
are collected by centrifugation, resuspended in .mu.l ml of L broth, and 
then incubated at 30.degree. C. for one hour. Aliquots of the cell mixture 
are plated on L-agar (L-broth with 15 g/L agar) plates containing 10 
.mu.g/ml tetracycline, and the plates are incubated at 30.degree. C. E. 
coli K12 RV308/pOGO216 transformants were verified by selection for 
tetracycline resistance and by restriction enzyme analysis of the plasmid 
DNA of the transformants. Plasmid DNA was obtained from the E. coli K12 
RV308/pOGO216 transformants for restriction enzyme analysis in substantial 
accordance with the teaching of Example 1B, but on a smaller scale, and 
the CsCl-gradient steps were omitted. 
B. Culture of E. coli K12 RV308/pOGO216 for Expression of Isopenicillin N 
Synthetase Activity 
Several isolates of the E. coli K12 RV308/pOGO216 transformants were 
individually inoculated into 5 ml aliquots of L broth containing 10 
.mu.g/ml tetracycline, and the cultures were incubated in an air-shaker 
incubator at 30.degree. C. until the O.D..sub.590 was .about.0.2 
absorbance units. The cultures were then transferred to a 42.degree. C. 
air-shaker incubator and incubated at 42.degree. C. for .about.6 hours. 
After the six-hour, 42.degree. C. incubation, one ml of each culture was 
collected, and the cells were pelleted by centrifugation. The cell pellets 
were individually washed with 1 ml of 10 mM NaCl and then resuspended in 
1.0 ml of IPNS extraction buffer (0.05 M Tris-HCl, pH=8.0; 0.01 M KCl; and 
0.01 M MgS04) The cells were sonicated by six, five-second bursts of 
sonication delivered by a Sonifier Cell Disruptor, Model W185, Heat 
Systems-Ultrasonics, Inc., Plainview, Long Island, NY, using the micro 
tip. The time between bursts of sonication was 60 seconds, and the mixture 
was kept in an ice-ethanol bath during the procedure. After sonication, 
the cell mixture was centrifuged to remove debris and then used directly 
in the assay. 
C. Assay for Isopenicillin N Synthetase Activity 
The following assay procedure is derived from the procedure of Shen et al., 
1984, J. of Antibiotics 37(9): 1044-1048. The isopenicillin N synthetase 
assay reaction was carried out in a total volume of 500 .mu.l. To start 
the reaction, 1.0 ml of a solution of 1.4 mM 
.delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine and 3.75 mM DTT was 
allowed to react at room temperature for 30-60 minutes to reduce any 
dimeric tripeptide to the monomeric form. Fifty .mu.l of each of the 
following stock solutions were aliquoted into each assay tube (sterile, 
glass, disposable 13.times.100 mm tubes): 500 mM Tris-HCl, pH=7.4; 100 mM 
KCl; 100 mM MgSO.sub.4 ; 2.0 mM FeSO.sub.4 ; and 6.7 mM ascorbic acid. 
Next, varying amounts of extract, diluted with water to a volume of 150 
.mu.l, were added. About 100 .mu.l aliquots of the tripeptide solution 
were then added to each tube; the addition of the tripeptide starts the 
reaction. Each tube was vortexed upon addition of the substrate. The 
reaction mixture vessels were then placed in a gyrotory shaker bath at 250 
rpm, with an incubation temperature of 25.degree. C. The reaction time was 
45 minutes. 
After 45 minutes of reaction, 2 samples of 100 .mu.l each were withdrawn 
and dispensed into wells in the bioassay plates, and 100 units of 
penicillinase A were added to the remainder of the sample. The 
penicillinase A is obtained from Riker's Laboratories, Inc.; the enzyme is 
sold in vials of 100,000 units, which are rehydrated to 5.0 mls with 
H.sub.2 O. Five .mu.l (100 units) of the rehydrated pencillinase A were 
added to the remainder of each reaction mixture, allowed to react for 5 
minutes at room temperature, and then 100 .mu.l of each penicillinase 
A-treated extract was dispensed into the wells of a bioassay plate. This 
penicillinase A treatment is done to check that the zones on the bioassay 
plate are due to the presence of a penicillin rather than a cephalosporin 
or other contaminant. 
The penicillin N standard curve was prepared by adding 0.5, 1.0, 2.0, 5.0, 
10.0, and 20.0 .mu.g of penicillin N to bioassay wells. The penicillinase 
A activity was also checked by adding 5 .mu.l of the enzyme preparation to 
.about.200 .mu.l of 0.2 .mu.g/ml penicillin N. 
The bioassay plates were composed of K131 nutrient agar, which is prepared 
by dissolving 30.5 g BBL Antibiotic Medium #11 (Becton Dickinson & Company 
Cockeysville, Md.) in 1 liter of deionized water, bringing the solution to 
a boil, cooling to 70.degree. C., and then autoclaving 35 minutes at 
121.degree. C. and 15 psi. The plates were seeded with 4 ml of a fresh 
overnight culture of Micrococcus luteus (ATCC 9341) per 700 ml of agar. 
The M. luteus was grown in K544 nutrient broth, which is composed of: 
Difco peptone, 5.0 g; Difco yeast extract, 1.5 g; sodium chloride, 3.5 g; 
dipotassium phosphate (anhydrous), 3.7 g; monopotassium phosphate, 1.3 g; 
Difco beef extract, 1.5 g, in 1 liter of deionized water--the solution is 
brought to a boil, cooled to 25.degree. C., adjusted to a pH=7.0 with 1 N 
HCl or 1 N NaOH, and then autoclaved for 20 minutes at 121.degree. C. and 
15 psi before use. The seeded agar was dispensed into 100.times.15 mm 
plates, at 15 mls of seeded agar per plate. The wells were prepared by 
applying suction using a disposable 15 ml pipette; each well was 10 mm in 
diameter. 
After the plates were prepared and the samples dispensed into the wells, 
the plates were placed in a 37.degree. C. incubator for 18 hours. The 
assay results were determined by measuring the diameter of the cleared 
areas around each sample well, which result from the M. luteus being 
unable to grow when a penicillin is present. 
The results of the assay demonstrate that the E. coli K12 RV308/pOG0216 
transformants express isopenicillin N synthetase activity.