Method to produce biotin

Details Method to produce biotin Method to produce biotin

1993-01-06

2001-08-21

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Preparing sulfur-containing organic compound

C435S252300, C435S252330, C435S320100, C435S471000, C435S476000, C536S023700

Reexamination Certificate

active

06277609

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method for producing biotin using recombinant cells transformed with nucleic acid sequences involved in biotin biosynthesis. In particular, this invention discloses a method to improve the ability of such recombinant cells to convert biotin vitamers to true biotin. The invention also discloses a method for improving over-all biotin production.
BACKGROUND OF THE INVENTION
Biotin, or vitamin H, is an indispensable element in intermediary metabolism in many organisms since it is an essential factor of biotin-dependent carboxylases important in fatty acid synthesis, gluconeogenesis, and amino acid metabolism. Biotin is useful as a food supplement, as a cosmetic additive, and as a diagnostic reagent in biotin-avidin-based detection assays.
Most biotin for commercial use is currently produced by a complex chemical synthesis process. Although several investigators are attempting to synthesize biotin in commercial quantities using microbiological methods, the cost thus far has been prohibitive. Wild type microorganisms produce only small amounts of the vitamin apparently because such microorganisms exert tight control over biotin biosynthesis. In an effort to improve microbial biotin production, some investigators have transformed microorganisms with
Escherichia coli
or
Bacillus sphaericus
genes that encode certain proteins involved in the biotin biosynthetic pathway. Although expression of these genes in some cases did increase true biotin and/or biotin vitamer production, the amount of true biotin produced using such methods is substantially lower than that required for a commercially viable process.
The biotin biosynthetic pathway in
Escherichia coli
is thought to include at least 5 enzymatic steps catalyzed by enzymes encoded by
Escherichia coli
bioA, bioB, bioF, bioC, and bioD genes contained on the biotin operon. The
Escherichia coli
bioA, bioB, bioD, and bioF genes are thought to encode enzymes having the following respective activities: 7,8-diaminopelargonic acid aminotransferase (also called 7,8-diaminopelargonic acid synthase), biotin synthetase (also called biotin synthase), desthiobiotin synthetase (also called desthiobiotin synthase), and 7-keto-8-aminopelargonic acid synthetase (also called 7-keto-8-aminopelargonic acid synthase). The protein encoded by the
Escherichia coli
bioC gene is thought to operate at an early step in the biotin biosynthetic pathway, but the protein's actual function is presently unknown. The biotin operon also includes an additional open reading frame, referred to as
Escherichia coli
ORF 1, the function of which, until the present invention, has been unknown (e.g., Otsuka et al., pp. 19577-19585, 1988,
J. Biol. Chem.,
vol. 263; Brown et al., pp. 295-326, 1991,
Biotech. Genet. Engineer. Reviews,
vol. 9; Eisenberg, pp. 544-550, 1987, in
Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology,
Neidhardt, F. C. et al., eds., American Society of Microbiology, Washington, D.C.). In addition, the
Escherichia coli
bioH gene, located at a site distant from the biotin operon, encodes a protein thought to be involved in an early, but as yet unknown, step in the biotin biosynthetic pathway (e.g., O'Regan et al., p. 8004, 1989,
Nucleic Acids Res.,
vol 17; Brown et al., ibid.
Two gene clusters encoding enzymes involved in biotin biosynthesis have been isolated from
Bacillus sphaericus.
The two gene clusters include the linked
Bacillus sphaericus
genes bioD, bioA, bioY, and bioB, also referred to as
Bacillus sphaericus
bioDAYB; and linked
Bacillus sphaericus
genes bioX, bioW, and bioF, also referred to as
Bacillus sphaericus
bioXWF (see, for example, Gloeckler et al., pp. 63-70, 1990,
Gene,
vol. 87; U.S. Pat. No. 5,096,823 by Gloeckler et al., issued Mar. 17, 1992; European Patent Office Publication No. 266,240, by Gloeckler et al., published May 4, 1988; and European Patent Publication No. 240,105, by Ohsawa et al., published Nov. 7, 1987).
Bacillus sphaericus
and
Escherichia coli
bioA, bioB, bioD, and bioF genes are structurally similar and apparently encode functionally equivalent enzymes (e.g., Brown et al., ibid.).
Bacillus sphaericus
bioW, bioX and bioY genes, which apparently are not structurally homologous to known
Escherichia coli
genes, are thought to be involved in the active uptake of pimelic acid by
Bacillus sphaericus
(e.g., Brown et al., ibid.). In contrast, some investigators have hypothesized that uptake of pimelic acid by
Escherichia coli
is by passive diffusion (e.g., Brown et al., ibid.; Ploux et al., pp. 685-690, 1992,
Biochem. J.,
vol. 287).
Several investigators have disclosed systems to attempt to express biotin using the
Escherichia coli
biotin operon. For example, GB Publication No. 2,216,530, by Pearson et al., published Oct. 11, 1989, discloses expression of the
Escherichia coli
biotin operon in
Saccharomyces cerevisiae
but does not report biotin production levels. In another example, Fisher, in U.S. Pat. No. 5,110,731, issued May 5, 1992, discloses that a biotin retention-deficient mutant of
Escherichia coli
transformed with a plasmid containing the
Escherichia coli
biotin operon produced a maximum of 30 milligrams (mg) of biotin per liter of medium.
Several researchers (see, for example, Ogata, pp. 390-394, 1970,
Methods in Enzymology,
vol. 17a; Izumi et al., pp. 231-256, in
Biotechnology of Vitamins, Pigments, and Growth Factors,
Elsevier Applied Science, E. J. Vandamme, ed.; U.S. Pat. No. 3,393,129, by Shibata et al., issued Jul. 16, 1968; and U.S. Pat. No. 4,563,426 by Yamada et al., issued Jan. 7, 1986) have reported that true biotin and biotin vitamer production by fungal and bacterial microorganisms, and in particular by
Bacillus sphaericus,
increases when the microorganisms are grown in the presence of biotin precursors, such as pimelic acid and desthiobiotin. Based upon this observation, attempts have been made to increase biotin production by transforming
Escherichia coli
and
Bacillus sphaericus
microorganisms with either the
Bacillus sphaericus
bioB gene or
Bacillus sphaericus
bioDAYB and bioXWF biotin gene clusters and growing the transformants in the presence of biotin precursors.
European Patent Publication No. 375,525, by Gloeckler et al., published Jun. 27, 1990, discloses the use of
Escherichia coli
host cells transformed with the two clusters of
Bacillus sphaericus
biotin operon genes (i.e., bioDAYB and bioXWF) to produce biotin. When such transformed hosts were grown in medium containing pimelic acid, they produced 144-160 mg of biotin vitamers per liter of medium but only 15-16 mg of true biotin per liter of medium. Thus, the amount of true biotin produced was only about 9 to 10 percent of the amount of total biotin (i.e., true biotin and vitamers) produced, indicating that, despite a high gene copy number, the transformed cells could not completely convert the biotin vitamers to true biotin. In addition, of the total amount of biotin vitamers produced, only 25 percent to 28 percent was desthiobiotin (the direct precursor of biotin), suggesting that about 70 percent of the biotin vitamers produced were compounds that had yet to be converted to desthiobiotin.
Sabatié et al., pp.29-50, 1991,
Journal of Biotechnology,
vol. 20, also transformed
Escherichia coli
cells with a vector containing the
Bacillus sphaericus
bioDAYB and bioXWF gene clusters. When such transformed cells were grown in the presence of pimelic acid under fed-batch fermentation conditions, the cells produced 300 mg of biotin vitamers per liter of medium, but only 45 mg of true biotin per liter of medium. Thus, the amount of true biotin produced by Sabatié et al. was only 13 percent of the total amount of biotin (i.e., true biotin and vitamers) produced, again indicating inefficient conversion of biotin vitamers to true biotin.
Ohsawa et al., pp. 39-48, 1989,
Gene,
vol. 80, transformed
Escherichia coli, Bacillus sphaericus
and
Bacillus subtilis
with vectors containing the
Bacillus

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