Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1996-09-24
1998-11-10
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435190, 4352523, 4353201, 435822, 536 232, C12P 760, C12N 904, C12N 1500, C12N 100
Patent
active
058342630
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for producing 2-keto-L-gulonic acid (hereinafter also referred to as 2KLGA), which is a precursor of L-ascorbic acid, efficiently and with ease by genetic engineering.
The present invention also relates to a series of expression systems involved in the efficient production of 2KLGA.
BACKGROUND ART
The 2KLGA is a key intermediate in the synthesis of L-ascorbic acid. For industrial production, 2KLGA is chemically synthesized from D-sorbitol by oxidation according to the Reichstein's method. Meanwhile, many microorganisms inclusive of the microorganisms belonging to the genus Gluconobacter are known to convert D-sorbitol to 2KLGA through an enzymatic oxidation. The microorganisms belonging to the genus Gluconobacter have been improved by genetic engineering using conjugal transfer and transposon. However, because of the low production of 2KLGA by these microorganisms, they have not been utilized in the industrial production yet.
Accordingly, there has been a desire for a more efficient and simplified method for the production of 2KLGA.
It is also well known that, in secondary metabolite production by microorganisms such as that of 2KLGA, a mere insertion of a gene (group) responsible for the biosynthesis of a substance into a plasmid and culture of the cells of microorganism, which have been recombined with this plasmid, does not necessarily result in an improved production of the et al., J. Gen. Microbiol., 137, pp. 2331-2337 (1991)!.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide an expression vector containing both a DNA encoding L-sorbose dehydrogenase (hereinafter SDH) and a DNA encoding L-sorbosone dehydrogenase (hereinafter SNDH), a transformant having an ability to produce 2KLGA at high yields from D-sorbitol, which has been transformed with said expression vector, and an efficient and simplified process for producing 2KLGA, which comprises culturing said transformant.
In an attempt to accomplish the above-mentioned objects, the present inventors have conducted intensive studies to succeed in obtaining an expression vector having the above-mentioned preferable property, and found that 2KLGA can be efficiently produced from D-sorbitol by a series of culture systems which comprise introducing said expression vector into a host microorganism capable of producing L-sorbose at high yields and having low 2KLGA-decomposing activity, or a host microorganism having, in addition to the above-mentioned properties, low L-idonic acid-producing activity, which resulted in the completion of the present invention.
Accordingly, the present invention relates to an expression vector containing both a DNA encoding SDH (hereinafter also referred to as SDH gene) and a DNA encoding SNDH (hereinafter also referred to as SNDH gene).
The present invention also relates to a microorganism capable of producing L-sorbose at high yields from D-sorbitol and having no or low 2KLGA-decomposing activity, or a host microorganism having, in addition to the above-mentioned properties, no or low L-idonic acid-producing activity. This microorganism is useful as a host into which the above-mentioned expression vector is introduced. The expression vector of the present invention includes not only that having an SDH gene and an SNDH gene on one vector, but also a pair of vectors separately having each gene.
The present invention further relates to a transformant which is a microorganism having the above-mentioned expression vector introduced therein, and which has an ability to produce 2KLGA at high yields from D-sorbitol.
In addition, the present invention relates to a process for producing 2KLGA, which comprises harvesting 2KLGA from a culture obtained by culturing said transformant in a medium containing D-sorbitol.
The present invention moreover relates to an expression vector, a host, a plasmid and a transformation method useful for efficiently and easily producing 2KLGA from D-sorbitol by genetic recombination.
BRIEF DESCRIPTION OF THE DRAWIN
REFERENCES:
patent: 4902617 (1990-02-01), Fujiwara et al.
patent: 4916069 (1990-04-01), Fujiwara et al.
patent: 5082785 (1992-01-01), Manning et al.
patent: 5399496 (1995-03-01), Fujiwara et al.
Balbas et al. (1990) Methods in Enzymology, vol. 185, pp. 14-37.
Hayashi Hiromi
Ishii Yoshinori
Niwa Mineo
Saito Yoshimasa
Yoshida Masaru
Fujisawa Pharmaceutical Co. Ltd.
Slobodyansky Elizabeth
Wax Robert A.
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