Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound other than saccharide containing a...
Reexamination Certificate
2000-08-23
2001-09-11
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound other than saccharide containing a...
C435S254100, C435S261000, C435S262000
Reexamination Certificate
active
06287800
ABSTRACT:
FIELD OF INVENTION
This invention relates to a new strain of
Gibberella fujikuroi
, LTB-1027 for the production of gibberellins, GA
3
, GA
4
and GA
7
. It further relates to a method of producing gibberellins GA
4
and GA
7
at high titer by the fermentation of strain LTB-1027 under controlled conditions.
BACKGROUND OF THE INVENTION
Gibberellins are a large family of closely related tetracyclic triterpenoid compounds first discovered as metabolites of an Ascomycete,
Gibberella fujikuroi
(perfect state of
Fusarium moniliforme
), which causes the bakanae disease of rice seedlings. The disease is typified by excessive stem and leaf elongation. Infected seedlings became abnormally tall and spindly and usually fall over. The culture filtrate of
Gibberella fujikuroi
produced a similar growth-promoting effect. From the filtrate a crystalline active product, later shown to be a mixture of gibberellins, was isolated. Gibberellins assumed a wider significance when it was discovered that gibberellins (many of which have not been detected in
G. fujikuroi
) are endogenous plant growth hormones.
One hundred and twenty-one gibberellins have been described (http://www.plant-hormones.bbsrc.ac.uk/gibberellin_information2.htm) and named gibberellin A
1
, A
2
, A
3
, A
4
, A
5
, . . . , approximately in the order they were discovered. Gibberellin A
3
(GA
3
), GA
4
, and GA
7
pertinent to this invention and their chemical structures are shown below.
Since gibberellins are endogenous plant hormones, their concentrations in plant tissues are quite low and are tightly regulated. Immature seeds, the best source of plant derived gibberellins, contain 10-100 &mgr;g per gram wet weight.
Gibberella fujikuroi
is by far the most abundant source of the gibberellins, and GA
3
is usually the major gibberellin component. Fermentation titers of GA
3
at 1,000 mg/liter have been achieved in commercial production, although the actual titer is purported to be a few times higher, and titers of 2,000 mg/liter have been claimed (Bruckner, B. & Blechschmidt, D.: The Gibberellin Fermentation, Critical Reviews in Biotechnology, 11(2), 163-192 (1991)).
Different gibberellins stimulate the growth of different parts of plants and are effective during different periods of a plant's growth cycle. Gibberellin A
3
primarily stimulates the growth of stems and leaves, while GA
4
and GA
7
primarily stimulate flowering and cause fruit cells to elongate. Mixtures of GA
4
and GA
7
(GA
4+7
) have been used successfully by growers of apples, pears and grapes to produce larger fruits and to achieve earlier harvests. The commercial production of GA
4
and GA
7
, however, has not been quite as successful as that of GA
3
, since GA
4
and GA
7
generally are minor metabolites of
Gibberella fujikuroi
. Titers of GA
4+7
at ~650 mg/liter with a GA
4
/GA
7
ratio of 1:5~6 have been reported previously (ICI Patent, EP 0112629B, 29.04.87). Furthermore, the separation of GA
4
and GA
7
from a mixture containing both is difficult and has not been economically feasible; thus, GA
4
and GA
7
are available commercially only as mixtures with GA
7
as the predominant component. As fine chemicals, the costs of GA
4
and GA
7
are approximately 300 times that of GA
3
.
Many other applications of GA
4+7
have been demonstrated and documented (Commercial uses of gibberellins, in
The Biochemistry and Physiology of Gibberellins
, Vol. 2, Crozier A., Ed., Praeger, New York, 1983). For example, the use of GA
4+7
for russet control of apples have demonstrated that the GA
3
in the GA
4+7
mixture caused reduction in flower bud formation and that GA
4
is most effective in russet control. GA
4
was found to be superior to GA
3
and GA
4+7
for promoting fruit set of many commercially grown apples. GA
4+7
was found to induce flowering in seed plants including many coniferous species (Pharis, R. P. & King, R. W.; Gibberellins & reproductive development in seed plants, Rev. Plant Physiol., 36, 517, 1985), as well as promote seed cone production (Ho, R. H., Gibberellin A
4+7
enhances seed cone production in field-grown black spruce, Can. J. For. Res., 18, 139, 1988), opening up the possibility of using GA
4+7
in forestry management.
A number of laboratories have searched for high producing strains of GA
4
and GA
7
that produce little or no GA
3
. Sphaceloma manihoticola
, a fungus that causes the super-elongation disease of cassava, produced GA
4
as the major gibberellin component (Graebbe, J. E., and Rademacher, W. B.; EP 0024951 B1). It also produced a number of other gibberellins, but no GA
3
and GA
7
. The fermentation titer of GA
4
, however, was only ~7 mg/liter and not of commercial importance. The laboratory of E. Cerda-Omedo reported a number of strains of
Gibberella fujikuroi
that produced GA
7
at the expense of GA
3
(Gibberellin Biosynthesis in gib mutants of
Gibberella fujikuroi
, J. Biol. Chem., 270:25, 14970-14974, 1995). The GA
7
titer, however, is only in the range of 60-80 mg/liter.
SUMMARY OF THE INVENTION
It is an object of this invention to employ a new mutant of
Gibberella fujikuroi
Strain LTB-1027 to produce mixtures of gibberellins that are high in the gibberellins GA
4
and GA
7
. Another object of this invention is to produce gibberellins GA
4
and GA
7
in much higher titers than have been produced in the past.
The method disclosed herein produces a mixture of gibberellins which is predominantly GA
4
and GA
7
but also contains GA
3
. The method has the following steps: a) providing a seed of
Gibberella fujikuroi
Strain LTB-1027 or mutants derived therefrom; b) inoculating the seed into a culture medium rich in carbohydrate and relatively low in nitrogen; c) incubating the culture for at least four days; d) separating the
Gibberella fujikuroi
Strain LTB-1027 from the culture broth; and e) extracting the gibberellins to produce a gibberellin mixture which is at least 50% GA
4
and GA
7
.
In a preferred embodiment, the gibberellin mixture is at least 70% GA
4
and GA
7
. In an even more preferred embodiment, the gibberellin mixture is at least 80% GA
4
and GA
7
.
In another embodiment, the method yields a gibberellin mixture with a combined titer of GA
4
and GA
7
in excess of 800 mg/liter.
In yet another embodiment, the method of claim I yields a gibberellin mixture with approximately equal titers of gibberellins GA
4
and GA
7
.
In a further embodiment, a disclosed method produces a gibberellin mixture which contains over 40% GA
4
.
DESCRIPTION OF THE INVENTION
This invention describes the production of gibberellins by a mutant strain of
Gibberella fujikuroi
. This strain is capable of producing more than 1,000 mg/liter of a mixture of gibberellins in which the ratio of GA
4+7
: GA
3
greater than 4:1 and there are approximately equal amounts of GA
4
and GA
7
. It is expected that under the appropriate fermentation conditions, this strain can produce higher titers of GA
4
at the expense of GA
7
. This strain was developed through repeated mutagenesis and strain selection starting from a
Gibberella fujikuroi
mutant that produced little GA
3
. This strain is maintained in the MicroFerm Culture Collection of Microcide Pharmaceuticals, Inc., 850 Maude Avenue, Mountain View, CA 94043 as culture number LTB-1027. A viable culture of this new
Gibberella fujikuroi
strain has been deposited on Oct. 19, 1999, under conditions of the Budapest Treaty with the Patent Culture Collection Laboratory, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 U.S.A. and has been added to its permanent collection. It has been assigned the strain designation NRRL 30227 by said depository.
Colonies of
Gibberella fujikuroi
LTB-1027 on Potato-Dextrose Agar (PDA) plates form aerial hyphae of the texture of cotton candy, 0.2-0.7 cm in height, white or light purple in color and orange-purple-brown substrate pigmentation.
Cultivation conditions of
Gallazzo Jorge L.
Lee May D.
Guttman Harry J.
Weber Jon P.
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