Mutants, blocked, production strain

Generation and selection of mutants played an essential role in the development of strains for industrial production of rifamyctns. In addition, auxotroffltic mutants and mutants blocked in the rifamycin biosynthetic pathway constituted the basic tools for genetic studies and the elucidation of biosynthetic steps. 

When the original A. medicerranei strain is cultured without addition of barbiturate to the medium, nfamycins A, C, D, and E (the so-called rifamycin complex) are produced, together with small amounts of rifamycin B. The chemistry and the biosynthesis of these compounds have never been properly investigated. However, cosynthesis experiments, in which a mutant blocked in an early step of the biosynthesis was grown together with a rifamycin S-producing strain, demonstrated that the members of rifamycin complex arc transformation products of rifamycin S (47). 

Microbiological Methods. The low substrate specificity of many of the enzymes involved in GA biosynthesis in Gibberella fujikuroi has been utilized for the preparation of higher plant GAs. Suitable analogs of the natural GA-precursors are converted by the fungus to the corresponding GA analogs. It is usual to prevent the synthesis of the natural GAs in order to facilitate purification of the unnatural products. A mutant strain, Bl- la, in which GA biosynthesis is blocked early in the pathway (67) (between ent-kaurenal and ent-kaurenoic acid) has been used. 

L-Camitine is used in infant health, sport and geriatric nutrition. The biotransformation is catalyzed by carnitine dehydratase in whole cells (Fig. 19-36). (R)-camitine is produced with > 99.5% conversion of butyrobetaine and > 99.5% ee. The mutant strain has blocked the L-carnitine dehydrogenase and excretes the accumulated product. The purified enzyme could not be used for the biotransformation because of its high instability. Apart from usual batch fermentations, continuous production... 

The generation from a producing organism of mutant strains that are blocked or altered in the biosynthetic pathway can lead to the isolation of related metabolites that could not otherwise be obtained. These metabolites may be intermediates from the blocked pathway that would normally be transient and detectable only in trace amounts, if at all, or they may represent shunt metabolites, whereby the intermediates have gone down a different biosynthetic route, resulting in novel compounds. The products of such mutants may be of interest in their own right, or they may be of interest in biosynthetic studies, biotransformation experiments or as starting points for precursor-directed biosynthesis. 

Production of L-aspartic acid from fumaric acid by stereoselective addition of ammonia under the action of the intracelluar aspartase in E. coli (Tanabe Seiyaku Co., Ltd.). When a 1000-liter column is used, theoretical yield of L-aspartic acid is 3.4 tons per day (and even considerably higher for mutant strains and plasmid pNKl01-harboring strains). A similar industrial process using the immobilized E. coli aspartase (instead of the whole cells) was established earlier by Kyowa Hakko Kogyo, Co., Ltd.. L-Aspartate is mainly used as a building block for the manufacture of the sweetener aspartame [170]. 

Another successful approach for blocking the intramolecular decomposition illustrated in Fig. 2 involved inhibition of the dehydration step leading to the anhydrohemiketal by replacement of the C-8 proton of erythromycin with fluorine. Preparation of flurithromycin (8-fluoroerythromycin, see Fig. 4) has been achieved by both chemical and biochemical methods. Addition of 8(S)-8-fluoroerythronolide A to a mutant strain of Streptomyces erythreus blocked in biosynthesis of its endogenous lactone (erythronolide) yielded the desired fluorinated erythromycin [40]. This technique of mutasynthesis has been further employed for the production of other fluorinated derivatives of erythromycin [40, 41]. Fluorination of different 8,9-anhydro-6,9-hemiketal derivatives of erythromycin by chemical means with reagents such as trifluoromethyl hypo-fluorite or perchloryl fluoride (with subsequent reduction of the N-oxide) has also been reported [42, 43]. 

Mutant strains of macrolide-producing microorganisms, in which different steps of macrolide biosynthesis have been blocked, have yielded a wide variety of new compounds representing biosynthetic intermediates and shunt metabolites. This approach has been employed especially with Streptomyces fradiae, producer of the commercially important veterinary antibiotic tylosin three groups have independently isolated and identified many fermentation products which differ from tylosin in the number or type of saccharides and/or degree of... 

Further improvement in production performance was achieved with a strain containing a tkt mutant allele (instead of the wild type) with drastically reduced enzymatic transketolase activity [180]. Transketolase is a key enzyme in the pentose phosphate pathway delivering ribulose-5-P and ribose-5-P, which are important building blocks for riboflavin biosynthesis. Presumably, the mutation impedes drainage of the pentose phosphate pathway intermediates back toward... 

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