Bacterial strains for the production of 2-keto-L-gulonic acid

Details Bacterial strains for the production of 2-keto-L-gulonic acid Bacterial strains for the production of 2-keto-L-gulonic acid

1999-09-10

2001-11-13

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S252100

Reexamination Certificate

active

06316231

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel bacterial strains useful for the production of 2-keto-L-gulonic acid. The present invention further relates to the use of these strains for the production of 2-keto-L-gulonic acid by fermentative conversion of L-sorbose. The present invention further relates to the use of these novel bacterial strains for the production of pyrroloquinoline quinone and a nontoxic lipopolysaccharide. Also described are the strains of the present invention transformed by a vector.
2. Background Information
2-Keto-L-gulonic acid (“2-KLG”) is a significant intermediate in the preparation of L-ascorbic acid (vitamin C), an essential nutrient. 2-KLG has been synthesized in the past on an industrial scale using the Reichstein method (
Helvetica Chimica Acta
7:311 (1934)). This method, however, has a number of disadvantages for commercial application, including the use of large quantities of solvents and the involvement of a number of complex reaction steps.
Accordingly, as an alternative to the Reichstein method, a number of processes employing one or more microorganisms have been developed to produce 2-KLG by fermentation. U.S. Pat. No. 2,421,611, for example, discloses a method involving microbial oxidation of D-glucose to 5-keto-D-gluconic acid, followed by chemical or microbial reduction to L-idonic acid and subsequent microbial oxidation to 2-KLG. Japanese Patent Publication Nos. 39-14493, 53-25033, 56-15877 and 59-35290, for example, disclose similar processes involving the microbial oxidation of D-glucose to 2,5-diketo-D-gluconic acid followed by microbial or chemical reduction to 2-KLG.
These methods, however, also suffer from a number of disadvantages that reduce their usefulness in commercial production of 2-KLG. For example, the chemical reduction steps in these methods (ie. the reduction of 5-keto-D-gluconic acid to L-idonic acid and 2,5-diketo-D-gluconic acid to 2-KLG) are accompanied by problems with controlling the stereochemistry of reduction (thus producing D-gluconic acid and 2-keto-D-gluconic acid, respectively, as byproducts) which, in turn, reduces the yield of 2-KLG. Alternatively, when this reduction is performed by one or more microorganisms, excess sugar is required to provide an energy source for the reduction, which also reduces the yield of 2-KLG.
In view of these problems, an alternate pathway has been employed for the fermentative production of 2-KLG, which involves only oxidation of L-sorbose to 2-KLG via a sorbosone intermediate. A number of processes have been developed using this pathway that employ a wide range of microorganisms from the genera Gluconobacter, such as
Gluconobacter oxydans
(U.S. Pat. Nos. 4,935,359; 4,960,695; 5,312,741; and 5,541,108), Pseudogluconobacter, such as
Pseudogluconobacter saccharoketogenes
(U.S. Pat. No. 4,877,735; European Patent No. 221 707), Pseudomonas, such as
Pseudomonas sorbosoxidans
(U.S. Pat. Nos. 4,933,289 and 4,892,823), and mixtures of microorganisms from these and other genera, such as Acetobacter, Bacillus, Serratia, Mycobacterium, and Streptomyces (U.S. Pat. Nos. 3,912,592; 3,907,639; and 3,234,105).
These processes, however, suffer from certain disadvantages that limit their usefulness for commercial production of 2-KLG. For example, the processes referenced above that employ
G. oxydans
also require the presence of an additional “helper” microbial strain, such as
Bacillus megaterium
, or commercially unattractive quantities of yeast or growth components derived from yeast in order to produce sufficiently high levels of 2-KLG for commercial use. Similarly, the processes that employ Pseudogluconobacter can require medium supplemented with expensive and unusual rare earth salts or the presence of a helper strain, such as
B. megaterium,
and/or the presence of yeast in order to achieve commercially suitable 2-KLG concentrations and efficient use of sorbose substrate. Other processes that employ
Pseudomonas sorbosoxidans
also include commercially unattractive quantities of yeast or yeast extract in the medium.
Pyrroloquinoline quinone (PQQ) (2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quino-line-4,5-dione) was initially isolated from cultures of methylotrophic (methanol-utilizing) bacteria and later was found to be present in many animal tissues. The structure of PQQ follows:
PQQ may be a novel vitamin as it is believed to be essential for normal growth and development. When fed to animals as a supplement, PQQ prevents oxidative changes that would ordinarily occur. Furthermore, PQQ increases nerve growth factor synthesis in mouse astrogial cells and has potential for a therapeutic role in the brain. (Bishop et al., “Pyrroloquinoline Quinone: A Novel Vitamin,”
Nutrition Reviews
56:287-293 (1998).
Organic chemical synthesis is the conventional means to produce PQQ. However, organic chemical synthesis has numerous disadvantages. For example, chemical synthesis is uneconomical and time consuming because the synthesis requires multiple and sometimes complicated reaction steps and produces low yields.
Accordingly, the need to overcome the disadvantages of chemical synthetic techniques for production of PQQ has been partially met by bacterial strains useful for the production of PQQ. (U.S. Pat. Nos. 4,994,382 and 5,344,768). However, there still remains a need for more efficient and economical PQQ-producing microorganism strains.
Lipopolysaccharide (LPS) is an amphipathic molecule which is a cell wall component of many gram-negative bacteria. It has been implicated in much of the pathophysiology associated with gram negative infections in humans and animals. LPS from
Rhodobacter sphaeroides
is non-toxic and has several uses as a immuno-modulator and antitumor agent. However, there are several disadvantages associated with producing nontoxic LPS through
Rhodobacter sphaeroides,
for example, the inconvenience of culturing phototrophically.
SUMMARY OF THE INVENTION
The present invention provides microorganism strains which efficiently produce 2-keto-L-gulonic acid.
Another embodiment of the present invention is directed to strains for production of 2-keto-L-gulonic acid in cooperation with helper strains.
A further embodiment of the present invention provides a method for producing PQQ.
Another embodiment of the present invention is to provide a method for producing a nontoxic LPS.
An additional embodiment of the present invention is to provide the bacterial strains of the present invention transformed by a vector, and a method for the transformation of the bacterial strains by a vector.
These and other embodiments are accomplished by the methods of the present invention, which, in a first embodiment, is directed to a culture of any of the microorganism strains ADM 291-19 (NRRL B-30035), ADM 62A-12A (NRRL B-30037N), ADM 266-13B (NRRL B-30036), or mutants thereof.
Other features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or may be learned by practice of the invention. These advantages of the invention will be realized and attained by the methods particularly pointed out in the written description and claims hereof.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.


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