Ascorbic acid intermediates and process enzymes

Methods for producing ascorbic acid using recombinant means comprising the transfer of genetic material by conjugation, a host cell lacking, entirely or to such an extent as not to be commercially useful, one or more enzymes in the metabolic path converting glucose to 2 keto-L-gulonic acid.

BACKGROUND 
This invention concerns aspects of a process for production of ascorbic 
acid. It specifically relates to purification of a useful protein, to 
production of proteins using recombinant techniques and to the use of such 
proteins in chemical conversions. More particularly, the invention relates 
to purification of and recombinant production of 2,5-diketogluconic acid 
(2,5-DKG) reductase and the use of the reductase so produced in converting 
2,5-DKG stereoselectively into 2-keto-L-gulonic acid (2-KLG), as well as 
to the production of a single recombinant organism capable of synthesizing 
2-KLG. The 2-KLG produced is a useful intermediate in the production of 
ascorbic acid (vitamin C). 
Ascorbic acid has become a major chemical product in the United States, and 
elsewhere in the world, due to its importance in health maintenance. While 
there may be some controversy over its efficacy in ameliorating the 
tendency of individuals to contract certain minor illnesses such as for 
example. the common cold, there is no doubt that it is essential for human 
beings to ingest required amounts of vitamin C. It has become a matter of 
concern in recent years that "natural" foods may not provide adequate 
amounts of vitamin C. Accordingly, there has developed a large demand for 
ascorbic acid, both as an additive to foods which are marketed to the 
consumer with supplemented levels of this vitamin, and as a direct vitamin 
supplement. Furthermore, ascorbic acid is an effective antioxidant and 
thus finds applications as a preservative both in nutritional and in other 
products. 
There are a number of processes available, some commercially viable, for 
the production of vitamin C. Several of these result in the preliminary 
production of 2-keto-L-gulonic acid (2-KLG) which can then be rather 
simply converted to ascorbic acid through acid or base catalyzed 
cyclization. Accordingly, 2-KLG has become, in itself a material of 
considerable economic and industrial importance. 
Means are presently available in the art to convert relatively plentiful 
ordinary metabolites, such as, for example, D-glucose, into 
2,5-diketogluconic acid (2,5-DKG) by processes involving the metabolism of 
prokaryotic microorganisms. See, for example, U.S. Pat. Nos. 3,790,444 
(Feb. 5, 1974); 3,998,697 (Dec. 21, 1976); and EPO Application Publication 
No. 0046284 published Feb. 24, 1982. The availability of this 2,5-DKG 
intermediate offers a starting material which is converted to the desired 
2-KLG only by the single step of a two electron reduction. The reduction 
can be effected chemically or catalyzed enzymatically. Various bacterial 
strains are known which are capable of effecting this reduction. Such 
strains are found in the genera Brevibacterium, Arthrobacter, Micrococcus, 
Staphylococcus, Pseudomonas, Bacillus, Citrobacter and Corynebacterium. 
See for example, U.S. Pat. Nos. 3,922,194 (Nov. 25, 1975), 4,245,049 (Jan. 
13, 1981) and 3,959,076 (May 25, 1976). Such strains have indeed been used 
to effect this reduction; however, use of such strains per se without 
enzyme purification does not permit certain alternative approaches 
available with the use of purified enzyme. Such a system would permit, for 
example, continuous production through immobilization of the enzyme on a 
solid support. Further, access to the genetic machinery to produce such an 
enzyme permits manipulation and localization to achieve production of the 
enzyme at a site most convenient for the conversion of 2,5-DKG. Most 
important among such loci is a site within the same organism which is 
capable of effecting the production of 2,5-DKG. Thus, a single organism 
would use its own machinery to manufacture the 2,5-DKG in situ and then 
using the 2,5-DKG reductase gene provided through recombinant technology 
produce the desired product. 
It is helpful to understand the context into which the present invention 
finds utility, by representing the process in terms of the relevant 
chemical conversions. An outline of a typical overall process for 
manufacture of ascorbic acid is shown in Reaction Scheme 1. 
##STR1## 
The process conveniently begins with a metabolite ordinarily used by a 
microorganism such as, for example, D-glucose as shown in Reaction Scheme 
1. Through enzymatic conversions, which may include the enzymes D-glucose 
dehydrogenase, D-gluconate dehydrogenase and 2-keto-D-gluconate 
dehydrogenase, the D-glucose undergoes a series of oxidative steps to give 
2,5-diketo-D-gluconic acid. It has been shown that this series of steps 
can be carried out in a single organism. (U.S. Pat. No. 3,790,444, EPO 
Appln. A20046284 [supra]); such organisms are, for example, of the genus 
Gluconobacter, Acetobacter or Erwinia. 
Alternate preparations of ascorbic acid have circumvented the 2,5-DKG 
intermediate by a combination of fermentative and chemical oxidations, and 
are clearly more cumbersome than the process shown. Typical of these is 
the Reichstein synthesis which utilizes diacetone-2-keto-L-gulonic acid as 
a precursor to 2-KLG. This intermediate is generated through a series of 
reductive and oxidative steps involving fermentation, hydrogenation, and, 
e.g., permanganate oxidation. Such a sequence is more complex than the 
reaction scheme shown above. The conversion of 2,5-DKG into 2-KLG can also 
be carried out enzymatically (U.S. Pat. Nos. 3,922,194; 3,959,076 [supra]; 
and 4,245,049 [Jan. 13, 1981]). 
Means are presently well known in the art to convert the resulting 2-KLG 
into ascorbic acid. This may be done either in the presence of dilute acid 
and heat or in a two-step process utilizing preliminary esterification in 
methanol, followed by lactonization in base. Effective procedures are 
described in Crawford, T. C., et al., Advances in Carbohydrate Chemistry 
and Biochemistry, 37, 79-155 (1980). These alternatives are 
straightforward and take advantage of the greater stability and shelf life 
of 2-KLG over ascorbic acid. Thus, it is more desirable and convenient to 
stockpile the 2-KLG intermediate for subsequent conversion to the desired 
final product than to synthesize the ascorbic acid directly. 
Because of the improvements of the present invention, alternate, superior 
means are available to effect certain aspects of this overall conversion. 
In one approach, because the enzyme responsible for the conversion of 
2,5-DKG into 2-KLG has been isolated and purified, the reduction step can 
be carried out under more controlled conditions, including those whereby 
the enzyme is immobilized and the solution substrates are fed continuously 
over the immobilized catalyst. In addition, the availability of 
recombinant techniques makes possible the production of large amounts of 
such enzyme available for ready purification. Further, recombinant 
techniques permit the coding sequences and necessary expression control 
mechanisms to be transferred into suitable host organisms with improved 
characteristics. Thus, simply focusing on the conversion of 2,5-DKG to 
2-KLG, three levels of improvement are attainable: (1) stricter control 
over variables; (2) availability of continuous processing; and (3) 
selection of host organism for the enzyme which has desirable qualities 
pertinent to the reduction reaction. 
The scope of improvement permitted by the effective cloning and expression 
of the 2,5-DKG reductase is, however even broader. Because of the 
availability of the appropriate genetic machinery, it is possible, as well 
as desirable, to transform an organism which is capable of producing the 
2,5-DKG with the gene encoding the reductase. Thus, the same organism can 
effect the entire process of converting, for example, glucose or other 
suitable ordinary metabolite into the stable, storable intermediate 2-KLG. 
SUMMARY OF THE INVENTION 
The present invention effects dramatic improvements in the process for 
converting a commonly available metabolite such as glucose into 2-KLG, a 
stable storable precursor for ascorbic acid. The pathway of the process 
described by the present invention encompasses the step of converting 
2,5-DKG into 2-KLG. The current processes for formation of the 2-KLG 
intermediate involve, at best, the deployment of at least two organisms or 
killed cultures thereof, do not permit regulation of the enzyme levels 
available, and are limited to batchwise processes. 
Another aspect of the invention is a process for the construction of a 
recombinant expression vector for the production of 2,5-DKG reductase. 
Still another aspect includes the expression vector so produced, cells and 
cell cultures transformed with it, and the product of such cells and cell 
cultures capable of effecting the reduction of 2,5-DKG stereospecifically 
to 2-KLG. Still another facet of this aspect of the invention is a process 
for converting 2,5-DKG to 2-KLG using recombinant reductase. 
Finally, the invention also relates to a process for converting glucose or 
other ordinary microbial metabolite into 2-KLG by fermentation by a single 
recombinant organism and thereafter to ascorbic acid. It also relates to 
the recombinant organism, and thereafter to ascorbic acid. It also relates 
to particular microorganisms that may be transformed using the process of 
conjugation. It also relates to the recombinant organism capable of 
carrying out the conversion of glucose or other ordinary metabolite to 
2-KLG. Such an organism is conveniently constructed by transferring to a 
host cell capable of effecting the conversion of the initial metabolite to 
2,5-DKG an expression vector encoding and capable of expressing the 
sequence for the 2,5-DKG reductase. Alternatively, such a recombinant 
organism is constructed by transforming an organism already producing the 
2,5-DKG reductase with vectors encoding the enzymes responsible for the 
oxidation of metabolite to 2,5-DKG. In either event, use of proper 
promoters and ribosome binding sites within the construction of the 
expression vectors permits the expression of the gene or genes for the 
enzyme(s) in the host cell.

DETAILED DESCRIPTION 
A. Definitions 
As used herein, "2,5-DKG reductase" refers to a protein which is capable of 
catalyzing the conversion of 2,5-DKG stereoselectively to 2-KLG. Many 
species have been found to contain the reductase particularly members of 
the coryneform group, including the genera Corynebacterium, 
Brevibacterium, and Arthrobacter; hence it appears by virtue of present 
knowledge that a preferred source for the enzyme is a member of the 
coryneform group. In the specific example herein, the particular form of 
this enzyme present in Corynebacterium was cloned and expressed. However, 
other bacterial species, such as, for example, those from the genera 
Brevibacterium, Arthrobacter, Micrococcus, Staphylococcus, Pseudomonas, 
Citrobacter and Bacillus are also known to synthesize an enzyme with the 
same activity as this enzyme. These genera are illustrative of potential 
sources for an enzyme similar to that present in Corynebacterium which may 
be available to catalyze this conversion. Alternate sources in addition to 
these naturally occurring ones in the prokaryotic kingdom may well be 
found. In addition, as the invention herein discloses and makes available 
the genetic sequence encoding such enzymes, modifications of the sequence 
which do not interfere with, and may, in fact, improve the performance of 
this enzyme are also available to those knowledgeable in the art. Such 
modifications and altered sequences are included in the definition of 
2,5-DKG reductase as used in this specification. In short, the term 
2,5-DKG reductase has a functional definition and refers to any enzyme 
which catalyzes the conversion of 2,5-DKG to 2-KLG. 
It is well understood in the art that many of the compounds discussed in 
the instant specification, such as proteins and the acidic derivatives of 
saccharides, may exist in variety of ionization states depending upon 
their surrounding media, if in solution, or out of the solutions from 
which they are prepared if in solid form. The use of a term such as, for 
example, gluconic acid, to designate such molecules is intended to include 
all ionization states of the organic molecule referred to. Thus, for 
example, both "D-gluconic acid" and "D-gluconate" refer to the same 
organic moiety, and are not intended to specify particular ionization 
states. It is well known that D-gluconic acid can exist in unionized form, 
or may be available as, for example, the sodium, potassium, or other salt. 
The ionized or unionized form in which the compound is pertinent to the 
disclosure will either be apparent from the context to one skilled in the 
art or will be irrelevant. Thus, the 2,5-DKG reductase protein itself may 
exist in a variety of ionization states depending on pH. All of these 
ionization states are encompassed by the term "2,5-DKG reductase." 
Similarly, "cells" and "cell cultures" are used interchangeably unless the 
context indicates otherwise. Gene transfer refers to a transfer or 
exchange of genetic material from one source to a recipient. There are 
three frequently used methods of gene transfer in bacteria: transformation 
(sometimes referred to as transfection), transduction and conjugation. 
Other gene transfer techniques are protoplast fusion and R' plasmids 
(Holloway, B. W., J. Bacteriol. 133:1078 [1978]). The transfer of a gene 
to a cell or to cells comprising a cell culture amounts to the same 
activity; it is a gene transfer process in which exogenously added DNA is 
taken up by a bacterium. A cell or microorganism receiving the transferred 
gene by one of the three types of gene transfer that now replicates and 
expresses the DNA so taken up is referred to as a recipient cell or 
microorganism. It is each bacterium that takes up the transferred 
material, frequently an expression vector, although it is a culture of 
bacteria that is treated with the genetic material. 
"Transduction" refers to a gene transfer process in which a bacterial virus 
propagating on one strain of bacterium, the donor, picks up genetic 
information and upon infection of another strain of bacterium, the 
recipient, sometimes causes a heritable change. A recipient cell that 
acquires a donor trait by this process is called a transductant. 
"Conjugation" refers to a gene transfer process in which one strain of 
bacterium, the donor, possessing a conjugative plasmid, makes physical 
contact with another strain of bacterium, the recipient, and transfers 
genetic material. Conjugative plasmids possess "tra" genes that typically 
specify and/or control: synthesis of appendages referred to as donor pili, 
which allow donor cells to make contact with recipient cells; synthesis 
and release of substances that minimize donor-donor matings; and, 
conjugational transfer of DNA commencing from a definable transfer origin 
site on the conjugative plasmid molecule. The recipient which acquires 
donor genetic information is called a transconjugant. Strains with readily 
selectable genetic markers such as nalidixic acid or rifampicin 
resistance, or special nutritional requirements, are used in order to 
isolate transconjugants. "Transformation" refers to a gene transfer 
process carried out by various methods such as by nuclear injection or 
calcium treatment using calcium chloride as described by Cohen, F. N. et 
al., Proc. Natl. Acad. Sci. U.S.A. 69:2110 (1972). 
"Expression vector" includes vectors which are capable of expressing DNA 
sequences contained therein where such sequences are operably linked to 
other sequences capable of effecting their expression. It is implied, 
although not explicitly stated, that expression vectors must be replicable 
in the host organisms either as episomes or as an integral part of a 
chromosomal DNA; clearly a lack of replication would render them 
effectively inoperable. In sum, "expression vector" is also given a 
functional definition. Generally, expression vectors of utility in 
recombinant techniques are often in the form of "plasmids," which term 
refers to circular double stranded DNA molecules which, in their vector 
form are not linked to the chromosomes. Other effective vectors commonly 
used are phage and non-circular DNA. In the present specification, 
"plasmid" and "vector" are often used interchangeably, however, the 
invention is intended to include such other forms of expression vectors 
which serve equivalent functions and which are, or subsequently become, 
known. 
"Recombinant cells" refers to cells into which vectors, constructed using 
recombinant DNA techniques, have been introduced using one of the methods 
described above. "Host" cells refers to such cells before the DNA has been 
transferred. In general, recombinant cells produce protein products 
encoded by such recombinant vectors which protein products would not 
ordinarily be produced; however, the definition also includes cells 
containing vectors encoding proteins which are, coincidentally, encoded in 
the bacterial chromosome or otherwise endogenously expressed in the 
recipient cell. The definition includes any cell which is producing the 
product of a xenogeneic sequence by virtue of recombinant techniques. 
"Ordinary metabolite" refers to such carbon sources as are commonly 
utilized by bacteria for growth. Examples of such metabolites are glucose, 
galactose, lactose, fructose or other carbohydrates which are readily 
available foodstuffs for such organisms. Such metabolites are defined 
herein to include enzymatic derivatives of such foodstuffs which are 
convertible into 2,5-diketo-D-gluconic acid. Such derivatives include 
D-gluconic acid, D-mannonic acid, L-gulonic acid, L-idonic acid, 
2-keto-D-gluconic acid, 5-keto-D-gluconic acid, and 5-keto-D-mannonic 
acid. 
B.1 Cloning and Expression of 2,5-DKG Reductase 
Both the availability of large amounts of purified 2,5-DKG reductase and 
its ability to be generated in situ in an organism which makes 2,5-DKG is 
greatly aided by the process of the invention which provides a means for 
cloning and expression of the gene for the reductase enzyme. The general 
procedure by which this is accomplished is summarized as follows, and a 
specific example of such procedures is outlined herein below in Example 1. 
The gene encoding 2,5-DKG reductase is cloned in either plasmid or phage 
vehicles from a genomic library created by partially digesting high 
molecular weight DNA from Corynebacterium or other suitable source using a 
restriction enzyme. For 2,5-DKG reductase, a suitable restriction enzyme 
is Sau 3A. Alternatively, a limited digest with a restriction enzyme 
having greater specificity, such as BamHI or PstI, may be used. The 
restriction digest is then ligated either to plasmid vectors replicable in 
suitable bacterial hosts, or to phage sequences capable of propagation in 
convenient bacterial cultures. The resulting plasmid and phage libraries 
are then screened using probes constructed based on the known partial 
sequence of the 2,5-DKG reductase protein (see European Patent Application 
No. 84304277.1). The efficiency of probe design may be improved by 
selecting for probe construction those codons which are known to be 
preferred by bacterial hosts. Identification of the desired clones from 
the plasmid and phage libraries is best effected by a set of probes such 
that the desired gene will hybridize to all of the probes under suitable 
stringency conditions and false positives from the use of only one probe 
eliminated. Upon recovery of colonies or phage successful in hybridizing 
with the oligonucleotides provided as probes, identity of the sequence 
with the desired gene is confirmed by direct sequencing of the DNA and by 
in vivo expression to yield the desired enzyme. 
The complete functional gene is ligated into a suitable expression vector 
containing a promoter and ribosome binding site operable in the host cell 
into which the coding sequence will be transformed. In the current state 
of the art, there are a number of promotion/control systems and suitable 
prokaryotic hosts available which are appropriate to the present 
invention. Similar hosts can be used both for cloning and for expression 
since prokaryotes are, in general, preferred for cloning of DNA sequences, 
and the method of 2-KLG production is most conveniently associated with 
such microbial systems. E. coli K12 strain 294 (ATCC No. 31446) is 
particularly useful as a cloning host. Other microbial strains which may 
be used include E. coli strains such as E. coli B, E. coli X1776 (ATCC No. 
31537) and E. coli DH-1 (ATCC No. 33849). For expression, the 
aforementioned strains, as well as E. coli W3110 (F.sup.-, .lambda..sup.-, 
prototrophic ATCC No. 27325), bacilli such as Bacillus subtilus, and other 
enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, 
and various Pseudomonas species may be used. A particularly preferred 
group of hosts includes those cultures which are capable of converting 
glucose or other commonly available metabolites to 2,5-DKG. Examples of 
such hosts are generally found among the genera Acetobacter, 
Gluconobacter, Acetomonas and Erwinia. The taxonomy and nomenclature of 
these genera are such that the same or similar strains are sometimes given 
different names. For example, Acetobacter cerinus used in the example 
below is also referred to as Gluconobacter cerinus. Examples of particular 
hosts include, but are not limited to, Erwinia herbicola ATCC No. 21998 
(also considered an Acetomonas albosesamae in U.S. Pat. No. 3,998,697); 
Acetobacter (Gluconobacter) oxydans subspecies melanozenes, IFO 3292, 3293 
ATCC No. 9937; Acetobacter (Gluconobacter) cerinus IFO 3263 IFO 3266; 
Gluconobacter rubiginosus, IFO 3244; Acetobacter Fragum ATCC No. 21409; 
Acetobacter (Acetomonas) suboxydans subspecies industrious ATCC No. 23776. 
In general, plasmid expression or cloning vectors or conjugative plasmids 
containing replication and control sequences which are derived from 
species compatible with the host cell are used in connection with these 
hosts. The vector ordinarily carries a replication site, as well as 
marking sequences which are capable of providing phenotypic selection in 
transformed cells. For example E. coli is typically transformed using 
pBR322, a plasmid derived from an E. coli strain (Bolivar, et al., Gene 2: 
95 (1977)). pBR322 contains genes for ampicillin and tetracycline 
resistance and thus provides easy means for identifying transformed cells. 
For use in expression, the pBR322 plasmid, or other microbial plasmid must 
also contain, or be modified to contain, promoters which can be used by 
the microbial organism for expression of its own proteins. Those promoters 
most commonly used in recombinant DNA construction include the 
.beta.-lactamase (penicillinase) and lactose promoter systems (Chang et 
al,, Nature, 275: 615 [1978]; Itakura et al., Science 198: 1056 [1977]; 
Goeddel et al., Nature 281: 544 [1979]) and a tryptophan (trp) promoter 
system (Goeddel et al., Nucleic Acids Res., 8: 4057 [1980]; EPO 
Application No. 0036776). While these are the most commonly used, other 
microbial promoters have been discovered and utilized, and details 
concerning their nucleotide sequences have been published, enabling a 
skilled worker to ligate them functionally in operable relationship to 
genes in transformation vectors (Siebenlist et al., Cell 20: 269 [1980]). 
By suitable cleavage and ligation DNA sequences encoding 2,5-DKG reductase 
can be included in the aforementioned vectors prepared as outlined above. 
Any unnecessary or inhibitory sequences may be deleted and the prokaryotic 
enzyme may then be purified; or the intact or broken cells used directly 
as catalysts; or alternatively, the host may be chosen so that once 
transformed it is capable of effecting the entire conversion of glucose or 
other suitable metabolite to the desired 2-KLG product. 
B.2 Conversion of Glucose or Other Metabolite to 2-KLG by a Single 
Recombinant Organism 
The availability of recombinant techniques to effect expression of enzymes 
in foreign hosts permits the achievement of the aspect of the invention 
which envisions production of 2-KLG in a single host organism from a 
readily available metabolite. This method has considerable advantage over 
presently used methods in that a single viable organism fermentation is 
substituted for two fermentations, and there is at least a partial balance 
of the oxidizing and reducing equivalents required for this conversion. At 
present there is no naturally occurring organism which is known to be 
capable of catalysis of this entire sequence of steps such that 2-KLG is 
produced in usable quantities. Certain wild type organisms may produce 
2-KLG however the level produced is not sufficient to be economically 
practical. It has been observed that wild type A. cerinus (IFO 3263) has 
its own cytoplasmic 2,5 DKG reductase enabling the organism to convert 
glucose to 2-KLG at a level of approximately 0.2 g/l under controlled 
conditions. Under the same controlled conditions and using the method of 
this invention, described below, a plasmid capable of expressing 2,5 DKG 
reductase can be transferred to A. cerinus (IFO 3263) using the process of 
conjugation, enabling the recipient A. cerinus to produce 2-KLG from 
glucose at a level of 4.5 g/l [see Example 5(c)(2)]. 
Organisms are however, known which effect the conversion of glucose or 
other ordinary metabolic substrate, such as, for example, galactose or 
fructose into 2,5-DKG. Another group of organisms is known which effects 
the conversion of the 2,5-DKG into 2-KLG, the latter conversion, of 
course, being catalyzed by a single enzyme within that organism, but 
utilizing the power of that organism to supply reducing equivalents. 
One approach to producing a single organism conversion that is included in 
this invention comprises construction of an expression vector for 2,5-DKG 
reductase as outlined above, and transfer of this vector by any of the 
gene transfer methods mentioned above such as transformation, transduction 
or conjugation, into cells which are capable of the initial conversion of 
ordinary metabolites into the 2,5-DKG substrate for this enzyme. As 
outlined in Example 1 below, this gene transfer results in a single 
organism 2-KLG factory. The details of the vector construction, gene 
transfer, and use of the resultant organism are described in the 
specification. 
An alternative approach is to clone the genes encoding the enzymes known to 
effect the conversion of glucose or other ordinary metabolite to 2,5-DKG 
from the organisms known to contain them, to construct expression vectors 
containing these cloned gene sequences, and to transfer such vectors to 
cells which normally produce the 2,5-DKG reductase. Examples of the 
enzymes effecting the conversion of an ordinary metabolite to 2,5-DKG are 
D-glucose dehydrogenase (Adachi, O. et al., Agric. Biol. Chem., 
44(2):301-308 [1980] Ameyama, M. et al., Agric. Biol. Chem. 45[4]:851-861 
[1981]), D-gluconate dehydrogenase (McIntire, W. et al., Biochem. J., 
231:651-654 [1985]; Shinagawa, E. et al., Agric. Biol. Chem. 40[3]:475-483 
[1976]; Shinagawa, E. et al., Agric. Biol. Chem. 42[5]:1055-1057 [1978]), 
5-keto-D-gluconate dehydrogenase and 2-keto-D-gluconate dehydrogenase 
(Shinagawa, E. et al., Agric. Biol. Chem., 45(5):1079-1085 [1981]). A 
third approach is to transfer to a neutral host the entire sequence of 
enzymes comprising the conversion of an ordinary metabolite to 2-KLG. This 
last approach offers the advantage of choice of host organism almost at 
will, for whatever desirable growth characteristics and nutritional 
requirements it may have. Thus, the use as host cells of organisms which 
have the heritage of a reasonable history of experience in their culture 
and growth, such as E. coli and Bacillus, confers the advantage of 
uniformity with other procedures involving bacterial production of enzymes 
or substrates. 
Once the organism capable of carrying out the conversion has been created, 
the process of the invention may be carried out in a variety of ways 
depending on the nature of the construction of the expression vectors for 
the recombinant enzymes and upon the growth characteristics of the host. 
Typically, the host organism will be grown under conditions which are 
favorable to production of large quantities of cells. When a large number 
of cells has accumulated, the promoter(s) supplied with the recombinant 
gene sequences either become or are already active, permitting the 
transcription and translation of the coding sequences. Upon suitable 
expression of these genes, and hence the presence of the desired catalytic 
quantities of enzyme, the starting material such as glucose, is added to 
the medium at a level of 1-500 g/L and the culture maintained at 
20.degree. C. to about 40.degree. C., preferably around 
25.degree.-37.degree. C. for 1-300 hours until conversion to 2-KLG is 
effected. The starting material concentration may be maintained at a 
constant level through continuous feed control, and the 2-KLG produced is 
recovered from the medium either batchwise or continuously by means known 
in the art. 
C. General Methods Emoloved in the Invention 
In the examples below, the following general procedures were used in 
connection with probe construction, screening, hybridization of probe to 
desired material and in vector construction. 
C.1 Probe Preparation 
Synthetic DNA probes were prepared by the method of Crea, R. and Horn, T., 
Nucleic Acids Res., 8: 2231 (1980) except that 
2,4,6-triisopropylbenzenesulfonyl-3-nitro-1,2,4-triazole (TPS-NT) was used 
as coupling agent (de Rooij, J. et al., Rec. Trav. Chim. Pays-Bas, 98: 537 
[1979]). 
C.2 Isolation of Plasmids, Cleavage with Restriction Enzymes 
Plasmids were isolated from the identified cultures using the cleared 
lysate method of Clewell, D. B. and Helinski, Biochemistry 9: 4428 (1970), 
incorporated herein by reference, and purified by column chromatography on 
Biorad A-50 Agarose. Smaller amounts (mini-preps) were prepared using the 
procedure of Birnboim, H. C. Nucleic Acids Research 7: 1513 (1979). 
Fragments of the cloned plasmids were prepared for sequencing by treating 
about 20 .mu.g of plasmids with 10 to 50 units of the appropriate 
restriction enzyme or sequence of restriction enzymes in approximately 600 
.mu.l solution containing the appropriate buffer for the restriction 
enzyme used or sequence of buffers; each enzyme incubation was at 
37.degree. C. for one hour. After incubation with each enzyme, protein was 
removed and nucleic acids recovered by phenol-chloroform extraction and 
ethanol precipitation. Alternatively, plasmids were fragmented by DNAase I 
digestion in the presence of MnCl.sub.2 (Anderson, S., Nucleic Acids Res. 
9, 3015 [1981]) or by sonication (Deininger, P. L., Analyt. Biochem. 129, 
216 [1983]). After cleavage, the preparation was treated for one hour at 
37.degree. C. with 10 units Klenow DNA polymerase or T4 DNA polymerase in 
100 .mu.l of Klenow buffer (50 mM KPi, pH 7.5, 7 mM MgCl.sub.2, 1 mM BME), 
containing 50 nmol dNTP. Protein was removed and nucleic acids recovered 
as above, and the nucleic acids suspended in 40 .mu.l of loading buffer 
for loading onto 6 percent polyacrylamide gel, as described above, for 
sizing. (Alternatively, fragments may be cloned directly into an M13 
vector ). 
DNA sequencing was performed by the dideoxynucleotide chain termination 
method (Sanger, F. et al. Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977)) 
after cloning the fragments in an M13-derived vector (Messing et al. 
Nucleic Acids Res. 9, 309 (1981)). 
C.3 Ligation Procedures 
DNA fragments, including cleaved expression plasmids were ligated by mixing 
the desired components (e.g. vector fragment cut from 0.25 .mu.g plasmid 
is mixed with insert cut from 1 .mu.g of plasmid in 20 .mu.l reaction 
mixture) which components were suitably end tailored to provide correct 
matching, with T4 DNA ligase. Approximately 10 units ligase were required 
for .mu.g quantities of vector and insert components. The resulting 
plasmid vectors were then cloned by transforming E. coli K12 strain 294 
(ATCC 31446) or DH-1 (ATCC 33849). The transformation and cloning, and 
selection of transformed colonies, were carried out as described below. 
Presence of the desired sequence was confirmed by isolation of the 
plasmids from selected colonies, and DNA sequencing as described below. 
The following examples serve to illustrate but not to limit the invention: 
EXAMPLE 1 
Recombinant 2,5-DKG Reductase 
A. Probe Design 
The Tm of the Corynebacterium sp. (ATCC 31090) DNA was measured and found 
to be 81.5.degree. C. in 7.5 mM sodium phosphate, 1 mM EDTA (pH 6.8). This 
corresponds to a G+C content of 71 percent, using Pseudomonas aeruginosa 
DNA (Tm=79.7.degree. C. G+C=67 percent) as a standard. Hence, in synthesis 
of the probes, those codons known to be prevalent in bacterial DNAs of 
high G+C content (Goug. M., et al., Nucleic Acids Res. 10: 7055 [1982]) 
were employed: phe TTC; lys, AAG; val, GTG; pro, CCG; ala, GCC; asp, GAC; 
gln, CAG; arg, CGC; glu, GAG; asn, AAC; ser, TCC; ile, ATC; leu, CTG; gly, 
GGC; tyr, TAC. The amino acid sequence of the 2,5-DKG reductase from 
Corynebacterium sp., (ATCC 31090) shows that the purified enzyme contains 
a single N-terminal sequence (.mu.=undetermined) 
##STR2## 
(See European Patent Application No. 84304277.1). This sequence and the 
above codons were used to construct suitable probes, using the method of 
Anderson and Kingston (Proc. Natl. Acad. Sci. USA 80: 6838 [1983]). Two 43 
mers were synthesized by the phosphotriester method of Crea, R. et al., 
Nucleic Acids Res., 2331 (1980): 
5'GGCCTCCTCCACGGCGCGCTGGGCGTCGGCCGGCGGCACCTTG3'; and, 
5'CTCCATCCCGCAGCTGGGCTACGGCGTGTTCAAGGTGCCGCCG3'. The oligonucleotide 
probes are phosphorylated with 100 .mu.Ci [.gamma.-.sup.32 P] ATP (5,000 
Ci/mmole, Amersham) and polynucleotide kinase (P-L Biochemicals). 
B. Construction of a Plasmid Genomic Library 
Genomic DNA was isolated from Corynebacterium sp. ATCC 31090 by the method 
of Schiller et al., Antimicro. Agents Chemotherapy 18: 814 (1980). Large 
fragments (&gt;100 kb) were purified by CsCl density gradient centrifugation 
and partially digested with Sau 3A. The digest was size fractionated by 
agarose gel electrophoresis into size classes of 1-2 kb, 2-3 kb, 3-4 kb, 
and 4-6 kb. A genomic library was prepared from each size class of DNA 
fragments using the vectors pBR322 and pACYC184 (Bolivar, F. et al., Gene 
2: 95 [1977]; Chang, A. C. Y., and Cohen, S. N., J. Bacteriol. 134: 1141 
[1978]) by cleavage of the BamHI site and insertion of the Sau3A fragments 
using T4 DNA ligase. The resulting plasmids were used to transform a 
recA.sup.- derivative of E. coli strain MM294. (ATCC 31446) or DH-1 (ATCC 
33849) employing the transformation protocol of D. Hanahan (J. Mol. Biol., 
166: 557 (1983)), incorporated herein by reference. Each genomic library 
contained 10.sup.4 -10.sup.5 independent recombinants. In an alternative 
procedure, additional plasmid libraries were prepared in pBR322 using 
BamHI fragments (size range 2.0-2.5 Kb) and Pst fragments (size range 
0.5-1.5 Kb) of Corynebacterium sp. (ATCC 31090) DNA. 
C. Screening of the Plasmid Library in E. coli 
The colonies were picked into microtiter dishes, incubated overnight, and 
stamped onto nitrocellulose filters (BA85) placed on LB plates containing 
ampicillin or chloramphenicol. The remaining portions of the colonies in 
the microtiter dishes were preserved by adding 25 .mu.l of 42 percent DMSO 
and storing at -20.degree. C. 
The transferred portions of the colonies were incubated for 8 to 9 hours at 
37.degree. C. and amplified by transferring the filters to plates 
containing 12.5 .mu.g/ml chloramphenicol or spectinomycin and incubating 
at 37.degree. C. overnight. 
The plasmid library in E. coli is screened by filter hybridization. The DNA 
is denatured and bound to duplicate filters as described by Itakura, K. et 
al., Nucleic Acids Res. 9: 879-894 (1981). The filters for hybridization 
are wetted in about 10 ml per filter of 5X SET, 5X Denhardt's solution and 
50 .mu.g/ml denatured salmon sperm DNA, and 0.1 percent sodium 
pyrophosphate+20 percent formamide (5X SET=50 mM Tris-HCl [pH 8.0]. 5 mM 
EDTA, 500 mM NaCl; 5X Denhardt's solution=0.1 percent bovine serum 
albumin, 0.1 percent polyvinylpyrrolidone, 0.1 percent Ficoll; see 
Denhardt, Biochem. Biophys. Res Comm. 23: 641 [1966]). The filters are 
prehybridized with continuous agitation at 42.degree. for 14-16 hrs, and 
probed with .about.1.times.10.sup.8 cpm of probe as prepared in 
subparagraph A of this Example at 42.degree. C. The filters that were 
hybridized with the probes of subparagraph A are washed with 
0.2.times.SSC. 0.1 percent SDS, 0.1 percent sodium pyrophosphate at 
42.degree. C. for 3.times.30 min. Each of the duplicate filters is blotted 
dry, mounted on cardboard and exposed to Kodak XR5 X-Ray film with Dupont 
Cronex 11R Xtra life Lightning-plus intensifying screens at -70.degree. C. 
for 4-24 hrs. 
Cells from positive colonies were grown up and plasmid DNA was isolated by 
the method of Clewell and Helinski (Proc. Natl. Acad. Sci. U.S.A. 62: 1159 
[1969]). DNA was fragmented with AluI and PstI and subcloned into the 
vectors M13mp8 and M13mp9 (Messing, J. and Viera, J., Gene 19:269 [1982]). 
Subclones that hybridized to the probes were sequenced using the dideoxy 
chain termination procedure (Sanger, F. et al., Proc. Natl. Acad. Sci. 
U.S.A. 74: 5463 [1977]) in order to verify that the DNA coded for the 
2,5-DKG reductase. Those clones which hybridized to the probes were 
arbitrarily labelled pDKGR2 and pDKGR9. pDKGR2 clones contained a 2.2 Kb 
BamHI fragment of the 2,5-DKG reductase gene pDKGR9 clones contained a 
0.88 Kb PstI fragment of the 2,5 DKG reductase gene. 
D. Construction of Expression Vectors for 2,5-DKG Reductase 
The 2,5-DKG reductase gene, accompanied by either its own or a synthetic 
ribosome binding site, is inserted `downstream` of the E. coli trp (or 
tac) promoter or the pACYC184 CAT promoter on expression plasmids, which 
also contain a tetracycline resistance gene or other selectable marker, 
and an origin of replication derived from plasmids ColEl. 15A, or RSF1010. 
Some constructs may contain, in addition, an active gene coding for E. 
coli trp repressor or the E. coli lac repressor which allows the 
expression of the 2,5-DKG reductase gene to be regulated by exogenously 
added indole acrylic acid or isopropylthio-.beta.-galactoside (IPTG). 
Various mutated versions of the above plasmids are also employed for the 
expression of 2,5-DKG reductase. 
A cloned 2.2 Kb BamHI fragment of Corynebacterium sp (ATCC 31090) DNA, 
containing a portion of the 2,5-DKG reductase gene, was isolated with the 
43-mer probes as described above. An 0.12 Kb PstI/BamHI fragment of this 
plasmid was further used as a probe to isolate an overlapping 0.88 Kb PstI 
fragment of Corynebacterium sp DNA, which contained the rest of the gene. 
As described in FIG. 2, pDKGR2 (containing the 2.2 Kb BamHI fragment) was 
digested with NcoI, treated with E. coli DNA polymerase I Klenow fragment 
and dNTPs to create flush-ended DNA, then further digested with BamHI to 
release an 0.87 Kb fragment; this fragment was purified by electrophoresis 
on low-melting-point agarose. The plasmid pDKGR9 (containing the 0.88 Kb 
PstI fragment) was digested with PstI and BamHI, and the resultant 0.76 Kb 
fragment similarly isolated on low-melting-point agarose. The 0.87 Kb 
NcoI/BamHI fragment and the 0.76 Kb BamHI/PstI fragment were then combined 
with SmaI/PstI - digested M13mp9 and ligated to yield an M13 recombinant 
("mit12") with a 1.6 Kb insert of Corynebacterium sp DNA containing the 
entire 2,5-DKG reductase gene (FIG. 2). 
To mit12 single-stranded DNA a "deletion primer" (sequence: 
ACGGCCAGTGAATTCTATGACAGTTCCCAGC) and AluI fragments of M13mp9 DNA were 
annealed. This template-primer combination was then treated with E. coli 
DNA polymerase Klenow fragment in the presence of dNTPs and T4 DNA ligase 
to create in vitro heteroduplex mit12 RF molecules, as described by 
Adelman et al., DNA 2: 183 (1983). These molecules were used to transform 
the host JM101 (Messing, J. Recomb. DNA Tech. Bull 2:43 [1979]), and 
recombinant phage incorporating the desired deletion were detected by 
plaque hybridization using the deletion primer as a probe (Adelman et al., 
DNA 2:183 [1983]). This construction was designated mit12.DELTA. (FIG. 3). 
The mit12.DELTA. RF DNA was digested with EcoRI and HindIII to yield a 1.5 
Kb fragment containing the 2,5-DKG reductase gene. The human growth 
hormone expression plasmid, pHGH207-1ptrp.DELTA.RI5' 
(pHGH207-1ptrp.DELTA.RI5. is a derivative of pHGH207-1 [de Boer et al., 
Proc. Natl. Acad. Sci. U.S.A. 80: 21 (1983)] in which the EcoRI site 
between the ampicillin resistance gene and the trp promoter has been 
deleted), was digested with EcoRI and PstI to yield a 1.0 Kb fragment 
containing the E. coli trp promoter. pBR322 was digested with PstI and 
HindIII to yield a 3.6 Kb fragment. These three fragments were ligated 
together to form an expression plasmid for 2,5-DKG reductase, designated 
pmit12.DELTA./trpl (FIG. 3). This plasmid was unable to confer 
tetracycline resistance on host cells. A plasmid that would encode a 
complete tetracycline resistance function was constructed as follows: 
pmit12.DELTA./trpl DNA was digested with HindIII, treated with E. coli DNA 
polymerase I Klenow fragment and dNTPs to produce flush ended DNA, then 
digested with SphI; the resultant 5.6 Kb fragment was purified by 
electrophoresis on low melting agarose. Similarly, pBR322 DNA was digested 
with EcoRI, treated with E. coli DNA polymerase I Klenow fragment and 
dNTPs, digested with SphI, and the resultant 0.56 Kb fragment purified on 
low melting agarose. The 5.6 Kb and 0.56 Kb fragments were then ligated 
together to yield a tetracycline-resistant 2,5-DKG reductase expression 
plasmid designated ptrpl-35 (FIG. 3). The DNA sequence of the trp 
promoter, synthetic ribosome binding site, and 2,5-DKG reductase gene 
contained on this plasmid is shown in FIG. 4. 
E. Production of Recombinant 2,5 DKG reductase by Erwinia herbicola 
Cells are prepared for transformation by the method of Lacy and Sparks, 
Phytopathological Society, 69: 1293-1297 (1979). Briefly a loop of 
suitable host cells, Erwinia herbicola (ATCC 21998), or E. coli MM294 
(ATCC 314646), is inoculated into 5 ml of LB medium and incubated at 
30.degree. C. for 14-16 hrs. A 1:100 dilution of this overnight growth is 
inoculated into LB, the culture grown to OD.sub.590 of 0.4, and the cells 
recovered by centrifugation at 4.degree. C. The pellet was resuspended in 
1/3 volume of 10 mM NaCl, again centrifuged at 4.degree. C., the pellet 
resuspended in an equal volume of 30 mM CaCl.sub.2, and after 60 minutes 
at 0.degree. C., the cells again centrifuged at 4.degree. C. The pellet is 
resuspended in 1/12 volume of 30 mM CaCl.sub.2 and stored at 4.degree. C. 
overnight. (Alternatively, cells may be resuspended in 30 mM CaCl.sub.2, 
15 percent glycerol, and stored at -70.degree. C.) 
Transformation is effected by incubation of 1 .mu.g plasmid in 0.2 ml of 
competent cells at 0.degree. C. for 2 hr followed by heating to 42.degree. 
C. for 1 min. 3 ml of LB broth is added and cells are allowed to recover 
for 4 hrs at 37.degree. C., then cells are plated on selective medium as 
described by Lacy and Sparks (supra). Successful transformants are grown 
on LB broth to a density of O.D.550=1.0, then centrifuged and resuspended 
in minimal medium in the presence of 0.2 percent glucose. IAA or IPTG is 
then added to the medium, and after 0.5-1.0 hrs the cells recovered by 
centrifugation and lysed by treatment with lysozyme and a detergent (Tween 
80). 
The supernatant is assayed for the presence of 2,5-DKG reductase and the 
results are shown in Table I. The proteins were analyzed by SDS 
polyacrylamide gel electrophoresis (FIG. 5). The protein band representing 
the recombinant 2,5-DKG reductase was identified immunologically using the 
Western blotting procedure (Tobin, H. et al., Proc. Natl. Acad. Sci. 
U.S.A. 76, 4350 [1979]). 
TABLE I 
______________________________________ 
2.5-DKG reductase activity 
Extract (.DELTA. Absorbance (340 nm) min..sup.-1 /100 
______________________________________ 
.mu.L) 
pBR322 transformed 
-0.087 
Erwinia herbicola 
(ATCC 21998) 
ptrpl-35 transformed 
-1.925 
Erwinia herbicola 
(ATCC 21998) 
______________________________________ 
EXAMPLE 2 
Production of 2-KLG by Contacting Recombinant Organism with 2,5-DKG 
Cells of ptrpl-35 transformed Erw. herbicola (ATCC 21998) were streaked 
from a frozen glycerol stock onto LB solid medium (10 g/L tryptone, 5 g/L 
yeast extract, 10 g/L NaCl, 15 g/L agar) containing 5 mg/L tetracycline, 
then incubated 48 hrs at 30.degree. C. A single colony was picked and used 
to inoculate 5 ml of LB liquid medium containing 5 mg/L tetracycline, and 
this was shaken in a test tube for 16 hrs at 30.degree. C. 1.0 ml of this 
culture was used to inoculate 100 ml LB liquid medium containing 5 mg/L 
tetracycline, and this was then shaken at 200 rpm for 16 hrs at 30.degree. 
C. Cells were harvested by centrifugation washed once in an equal volume 
of fresh LB medium, then resuspended in 50 ml of LB medium containing 5 
mg/L tetracycline and 20 g/L 2,5-DKG. A 10 ml aliquot of this was shaken 
in a 125 ml flask at 200 rpm for 16 hrs at 30.degree. C. The resultant 
broth was analyzed by HPLC and found to contain 2.0 g/L of 2-KLG. A 
control culture containing pBR322-transformed Erw. herbicola (ATCC 21998), 
treated in a similar fashion, contained no 2-KLG. 
EXAMPLE 3 
Production of 2-KLG from Glucose by Recombinant Organism 
Cells of ptrpl-35-transformed Erw. herbicola (ATCC 21998) were streaked 
from a frozen glycerol stock onto LB solid medium (10 g/L tryptone, 5 g/L 
yeast extract, 10 g/L NaCl, 15 g/L agar) containing 5 mg/L tetracycline, 
then incubated for 48 hrs at 30.degree. C. A single colony was picked and 
used to inoculate 5 ml of LB liquid medium containing 5 mg/L tetracycline, 
and this was shaken in a test tube for 16 hrs at 30.degree. C. 1.0 ml of 
this culture was used to inoculate 100 ml of ATCC medium 1038 (3.0 g/L 
glucose, 5.0 g/L yeast extract, 5.0 g/L peptone, 7.5 g/L CaCO.sub.3, pH 
7.0) containing 5 mg/L tetracycline, and this was shaken at 200 rpm in a 
500 ml flask at 30.degree. C. for 16 hrs. Cells from 75 ml of this culture 
were then harvested by centrifugation, resuspended in 50 ml of fresh ATCC 
medium 1038 containing 5 mg/L tetracycline and 20 g/L glycerol, and shaken 
at 200 rpm in a 500 ml flask at 30.degree. C. for 48 hrs. The resultant 
broth was analyzed by HPLC and GC/MS and found to contain 1.0 g/L 2-KLG. A 
control culture containing pBR322-transformed Erw. herbicola (ATCC 21998), 
treated in a similar fashion, contained no 2-KLG. 
EXAMPLE 4 
Production of 2-KLG by Erwinia herbicola in Different Media 
The following experiment was performed to demonstrate the production of 
2-KLG from readily available carbon sources. 
The production of 2-KLG by Erwinia herbicola (ATCC 21998)/ptrpl-35 was 
accomplished by growing the cells in a medium containing the following: 
______________________________________ 
Yeast Extract (Nestles) 
10 g/l 
Calcium Carbonate 20 g/l 
Corn Steep Liquor 10 g/l 
Glucose 20 g/l 
Tetracycline 5 mg/l 
______________________________________ 
The glucose and the tetracycline were sterilized separately and added prior 
to inoculation. 
An inoculum was prepared by adding 1.0 ml of a frozen stock culture to 50 
ml of Luria broth containing 5 g/l glucose and 5 mg/l tetracycline. The 
250 ml baffled flask containing the inoculum was incubated at 30.degree. 
C. for 12 hours with shaking. 
A 250 ml baffled flask was filled with 50 ml of the production medium above 
and inoculated with 1 ml of the inoculum The flask was incubated with 
shaking at 30.degree. C. The pH of the medium at the time of inoculation 
was 5.1 due to the acidity of corn steep liquor. After 57 hours the pH had 
risen to 8.71 and 2-KLG was shown to be present at a concentration of 0.6 
mg/ml by HPLC. The presence of 2-KLG was confirmed by HPLC and GC-Mass 
Spectrometry. 
EXAMPLE 5 
Production of Recombinant 2,5 DKG reductase by Acetobacter cerinus 
Conjugal transfer of RP4::Mu from E. coli to Acetobacter suboxydans was 
reported by Murooka, Y. et al., J. Bacteriol. 145(1):358 (1981). 
Triparental mating of E. coli QSR39, E. coli 294 and Acetobacter cerinus 
was used to transfer the foreign plasmid DNA. Inoue, T. et al., J. 
Ferment. Tech. 63(1):1-4 (1985) subsequent to this invention describe a 
similar method. Conjugal transfer or mating was used to improve the 
efficiency of transformation of Acetobacter cerinus. Conjugal transfer 
efficiency has varied with various acetic acid bacteria. (See Murooka et 
al., supra and Inoue et al., supra.) CaCl.sub.2 -mediated transformation 
has also been used to introduce plasmid DNA into Acetobacter aceti. 
(Okumara, H. et al., Agric. Biol. Chem. 49[4]:1011 [1985]; Fukaya, M. et 
al., Agric. Biol. Chem. 49[7]:2083 [1985]; and Fukaya, M. et al., Agric. 
Biol. Chem. 49[7]:2091 [1985]). 
E. coli QSR39 (lei.sup.-, Bl.sup.-, R.sup.- M.sup.-, SuP.sup.+, Tp.sup.r, 
Tra.sup.+) containing the Inc P-1 plasmid R751 (Tp, Tra.sup.+) was used. 
(Meyer, R. J. and Shapiro, J. A., J. Bacteriol. 143: 1362-1373 [1980] and 
Johanpatia, R. S. and Dotta, N., J. Med. Microbiol. 7:169 [1974]). Also a 
nalidixic acid resistant strain of Acetobacter cerinus (IFO 3263) was 
isolated by plating 0.1 ml of a saturated culture of Acetobacter cerinus 
on mannitol plates containing 20 .gamma./ml nalidixic acid. 
A. Plasmid Constructions 
(1) R751 is a 50 Kb plasmid containing tra (transfer) gene. The tra gene 
products permit conjugal transfer of the 143 plasmid. R751 has been 
described by Meyer, R. J. and Shapiro, J. A., J. Bacteriol. 143: 1362-1373 
(1980). 
(2) Plasmid 143 contains the E. coli trp promoter, a synthetic ribosome 
binding site, and the Corynebacterium 2,5-DKG reductase gene and the 
tetracycline resistance gene from plasmid pBR322 inserted at the EcoRI 
site of plasmid RSF1010 (Bagdasarian et al., Gene 16:237-247 [1981] and 
Guerry, P. et al., J. Bacteriol. 117:619 [1974]). 
(3) RSF 1010 is a broad host range plasmid (Inc Q) able to replicate in a 
wide range of gram.negative bacteria. Plasmid RSF 1010 was digested with 
EcoRI. After the digestion was complete the EcoRI site was filled in by 
treating the DNA with 5 mM dNTPs and the Klenow fragment of Pol I. Plasmid 
ptrpl-35 was digested with ScaI and PvuII to produce a blunt-ended linear 
DNA fragment containing the Corynebacterium 2,5-DKG reductase gene under 
the control of the E. coli trp promoter and the tetracycline resistance 
gene from pBR322 under the control of its own promoter. Both digested DNAs 
were extracted with phenol/chloroform, ethanol precipitated, and 
resuspended in H.sub.2 O. The fragments were ligated together using T4 DNA 
ligase and used to transform E. coli strain MM294. Plasmid 143 was 
isolated and the presence of the correct gene sequences verified by 
restriction analysis and Southern blots (FIG. 6). 
B. Bacterial Matinzs 
Acetobacter cerinus (nal.sup.r strain, isolated as described above) was 
grown in mannitol broth (25 g/L mannitol, 5 g/L yeast extract, 3 g/L 
peptone). E. coli QSR39 (containing plasmid pR751) was grown in LB broth 
containing 2.5 .mu.g/ml trimethoprin. E. coli MM294 (containing plasmid 
p143) was grown in LB broth containing 5 .mu.g/ml tetracycline. 
0.5 ml of 0.6 OD.sub.590 cultures of Acetobacter cerinus, E. coli MM294 
(containing plasmid 143), and E. coli QSR39 (containing plasmid R751) were 
mixed and filtered onto a pre-moistened nitrocellulose membrane (0.45 
.mu.m pore size). The filter was incubated face-up on LB solid agar 
overnight at 27.degree. C. 
The filter was then placed in a sterile tube and its surface was rinsed 
with 1.0 ml mannitol broth with 10 .gamma./ml tetracycline and 10 
.gamma./ml nalixidic acid. 100 .mu.l, 10 .mu.l and 0.5 .mu.l were plated 
on mannitol solid agar plates containing 10 .gamma./ml tetracycline and 10 
.mu.g/ml nalidixic acid. The plates were incubated at Z7.degree. C. 
overnight. 
Colonies that appeared on the plates were restreaked on YGC (50 g/L 
glucose, 5 g/L yeast extract, 12.5 g/L CaCO.sub.3) solid agar plates 
containing 10 .gamma./ml tetracycline. Acetobacter cerinus transconjugants 
were identified as those that were tetracycline resistant and 
acid-producing (as determined by clear halo production). One of these, 
termed Acetobacter cerinus (p147), was chosen for further 
characterization. 
A plasmid prep from Acetobacter cerinus (p147) was prepared and analyzed by 
restriction mapping and Southern blotting. This analysis showed that 
Acetobacter cerinus (p147) contained an RSF1010-based plasmid encoding the 
2,5-DKG reductase gene that was indistinguishable from plasmid 143, 
indicating that plasmid transfer from E. coli had occurred. 
The supernatant was assayed for the presence of 2,5-DKG reductase using 
methods known to the ordinarily skilled artisan and the proteins analyzed 
by SDS polyacrylamide gel electrophoresis (FIG. 7). The protein band 
representing the recombinant 2,5-DKG reductase was identified 
immunologically using the Western blotting procedure (Tobin, H. et al., 
Proc. Natl. Acad. Sci. U.S.A. 76: 4350 [1979]). 
An alternative mating procedure that did not require preselection of an 
Acetobacter cerinus IFO3263 nalidixic acid-resistant strain was also used. 
This procedure takes advantage of the vitamin B1 requirement of the p143 
E. coli host strain, MM294, and the fact that Acetobacter cerinus does not 
require vitamin B.sub.1. The procedure is identical to that described 
above, except that after the mating has been allowed to proceed on the 
filter, the cells are plated on minimal glucose or mannitol plates (5 g/L 
glucose or mannitol 6 g/L Na.sub.2 HPO.sub.4, 3 g/L KH.sub.2 PO.sub.4, 0.5 
g/L NaCl, 1 g/L NH.sub.4 Cl, 1 mM MgSO.sub.4, 1 mM CaCl.sub.2) containing 
0.25 mM nicotinamide and 15 .mu.g/ml tetracycline in order to select for 
Acetobacter cerinus (p147) transconjugants. 
C. Fermentation of Glucose to 2-KLG by A. cerinus IFO 3263/p147 
C.1 Preparation of the Inoculum 
A. cerinus 3263 IFO/p147 was cultivated in an aqueous medium containing a 
carbon source, such as glucose, glycerol, citrate, etc. at a concentration 
from 1 to 20 g/L. Nitrogen was supplied as inorganic ammonium salts such 
as ammonium sulphate or ammonium chloride usually in the range of 0.05 to 
1 g/L. Alternatively organic nitrogen such as yeast extract, hydrolyzed 
casein, or urea was used. Minerals were supplied. In many cases minerals 
contained in an organic nitrogen source such as yeast extract or casamino 
acids, were sufficient for growth. However, additional magnesium, 
phosphorous, potassium were included in the medium in the form of salts 
such as potassium chloride, potassium phosphate, magnesium sulphate or 
similar salts, generally in amounts from 0.1 to 2.0 grams per liter of 
medium. Tetracycline HCL was added to the medium in a concentration of 5 
to 40 mg/L. 
Generally, the Acetobacter strain was introduced into a medium containing 
the components described above. The microorganism was cultivated for a 
period of 8 to 24 hours at a temperature of from 25.degree. to 35.degree. 
C. Oxygen was provided to the inoculum vessel either by shaking or by 
mechanical agitation and air sparging. The pH was maintained between 5.0 
and 7.5 preferably 5.5 to 6.5, with a suitable buffer such as CaCO.sub.3, 
MES (2-(N-Morpholino)-ethanesulphonic acid), or potassium phosphate. 
Alternatively, the pH could be controlled by addition of acid such as 
phosphoric or sulfuric and base such as potassium hydroxide or sodium 
hydroxide. 
C.2 Preparation of the Production Medium and Production of 2 KLG from 
Glucose 
A suitable volume, 0.1 to 10%, of Acetobactor cerinus inoculum culture is 
then added to a medium described as the production medium. Glucose is 
added to the initial culture medium in the concentration of 5 to 20 g/L. 
Alternatively a small amount of glucose, 2 to 5 g/L, can be added to the 
initial medium as a substrate for growth and the remainder can be added at 
a later time, 12 to 36 hours after inoculation, for production of 2-KLG. 
This medium should contain an organic nitrogen source, such as yeast 
extract in the concentration of 0.5 to 2 g/L as well as an inorganic 
nitrogen source such as ammonium sulphate in the concentration of 0.1 to 
0.5 g/L. Inorganic elements must also be included in the form of salts as 
was described for the inoculation medium. Tetracycline HCL was added to 
the medium at a concentration of from 5 to 20 mg/L. The medium was 
sterilized using either filtration or autoclaving. Those components which 
are not stable at autoclaving temperatures such as glucose, MES and 
tetracycline should be sterilized by filtration and added to the medium 
separately. 
The microorganism was cultivated for a period of 36 to 120 hours at a 
temperature of from 25.degree. to 35.degree. C. Oxygen was provided to the 
inoculum vessel either by shaking or by mechanical agitation and air 
sparging. The pH was maintained between 5.0 and 7.5, preferably 5.5 to 
6.5. A buffer was selected which did not interfere with 2-KLG production 
and which maintained the pH between 4 5 and 7.0, preferably 5.5 to 6.5. 
Buffers described for the inoculation medium were suitable for the 
production medium as well but other production buffers are also possible. 
Alternatively, pH can be controlled by addition of acid such as phosphoric 
or sulfuric and base such as potassium hydroxide or sodium hydroxide. 
The production of 2-KLG was observed using analytical methods previously 
described. 
The following medium was prepared for cultivation of the inoculum: 
______________________________________ 
Bacto Tryptone 10 g/l 
Bacto Yeast Extract 5 g/l 
NaCl 10 g/l 
______________________________________ 
pH was adjusted to 7.0 using NaOH and HCl. 
Volume was raised to 1 L with deionized water and autoclaved for 30 min, 
121.degree. C. 
A 250 ml baffled erlenmeyer flask containing 30 ml of Luria broth was 
inoculated with Acetobacter cerinus 3263 IFO/p147. The culture was 
incubated on a rotary shaker (250 rpm, 1.5 inch throw) for 12 hours at 
30.degree. C. After this time 0.1 ml of culture was inoculated into the 
following production medium. 
______________________________________ 
(NH.sub.4).sub.2 SO.sub.4 
0.4 g/l 
K.sub.2 HPO.sub.4 6.0 g/l 
KH.sub.2 PO.sub.4 4.0 g/l 
MgSO.sub.4.7 H.sub.2 O 1.0 g/l 
MES (2-[N-Morpholino]- 13.1 g/l 
ethanesulfonic acid) 
______________________________________ 
These salts were added to distilled water and sterilized by filtration. 
______________________________________ 
Yeast Extract 1.0 g/l 
Glucose 5.0 g/l 
Tetracycline HCl 
20 mg/l 
______________________________________ 
The pH was adjusted to 7.0 with NaOH. The components Difco Yeast Extract 
1.0 g/l and Glucose 5.0 g/l were sterilized separately by autoclaving at 
121.degree. C. for 30 min. They were added to the salts medium after all 
components had cooled to room temperature. After 24 hours 10 g/l 
additional glucose was added to the fermentation broth. 
The glucose was converted to 2-KLG (3.2 to 6.5 g/l) in an overall molar 
yield of from about 20 to 40% after 96 hours. Cell density reached from 1 
to 5 A.sub.550. The pH of the medium was reduced to between pH 5.0 to pH 
6.5, typically pH 6.0.