Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Patent
1998-05-01
1999-11-02
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
4351721, 435232, 4353201, 530 232, C12P 2500, C12N 1560, C12N 1531
Patent
active
059768444
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a process for preparing riboflavin using microorganisms having an altered isocitrate lyase activity.
Vitamin B.sub.2, also termed riboflavin, is essential for humans and animals. Vitamin B.sub.2 deficiency results in inflammation of the mucous membranes of the mouth and throat, cracks in the corners of the mouth, itching and inflammation in skin folds and similar skin damage, conjunctivitis, diminished visual acuity and clouding of the cornea. Cessation of growth and decrease in weight can occur in babies and children. Vitamin B.sub.2 is therefore of particular economic importance as a vitamin preparation for use in vitamin deficiency and as a feed additive. In addition, it is also employed as a food dye, for example in mayonnaise, ice cream, desserts such as blancmange, etc.
Riboflavin is prepared either chemically or microbially. In the chemical methods of preparation, the riboflavin is usually obtained as a pure end product in multistep processes, with it being necessary to employ relatively expensive starting compounds such as D-ribose.
The preparation of riboflavin using microorganisms provides an alternative to the chemical preparation of this compound. Renewable raw materials, such as vegetable oils, can be employed as starting compounds for the microbial synthesis.
The preparation of riboflavin by means of fermenting fungi such as Ashbya gossypii or Eremothecium ashbyii has been disclosed (The Merck Index, Windholz et al., eds. Merck & Co., page 1183, 1983); however, yeasts, such as Candida or Saccharomyces, and bacteria, such as Clostridium, are also suitable for producing riboflavin. Bacterial strains which overproduce riboflavin are described, for example, in EP 405370, in which document the strains were obtained by transforming the riboflavin biosynthesis genes from Bacillus subtilis. However, these procaryotic genes are unsuitable for a recombinant method for preparing riboflavin which uses eucaryotes such as Saccharomyces cerevisiae or Ashbya gossypii.
WO 93/03183 describes the cloning of the riboflavin biosynthesis-specific genes from the eucaryotic organism Saccharomyces cerevisiae. These genes can be used to construct recombinant eucaryotic microorganisms which enable riboflavin to be produced efficiently.
Frequently, however, the starting compounds and substrates of the riboflavin biosynthesis enzymes are only present in limited quantity in the microorganism, so that no increase in riboflavin production is achieved despite increasing riboflavin biosynthesis activity.
It is therefore an object of the present invention to provide an improved microbial process for producing riboflavin, which process uses microorganisms which have no substrate limitation, or only relatively slight substrate limitation, and consequently enable riboflavin to be produced at an increased rate.
We have found that this object is achieved, according to the invention, by the endogenous isocitrate lyase activity of the microorganisms employed being altered. The alteration can be determined in relation to the unaltered starting strain. There are a large number of options for obtaining such microorganisms which have an altered ICL activity.
One option is to alter the endogenous ICL gene in such a way that it encodes an enzyme which has an increased ICL activity in relation to the starting enzyme. An increase in the activity of the enzyme can be achieved, for example, by increasing substrate turnover by means of altering the catalytic center, or by abolishing the effect of enzyme inhibitors. An increase in enzyme activity can also be brought about by increasing synthesis of the enzyme, for example by means of gene amplification or by means of eliminating factors which repress enzyme biosynthesis. The endogenous ICL activity is preferably increased by mutating the endogenous ICL gene. Mutations of this nature can be generated either in a random manner using standard methods, such as by using UV irradiation or mutation-inducing chemicals, or in a specific manner using genetic engineering me
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Boddecker Theo
Kasler Bruno
Sahm Hermann
Schmidt Georg
Seulberger Harald
BASF - Aktiengesellschaft
Mayhew Bradley S.
Wax Robert A.
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