Mutagenesis For Overproduction Of Metabolites

Increased yields of microbially produced food additives can often be achieved by the selection of overproducing mutants. Such desirable mutants will frequently be produced spontaneously or by a mutagenic agent. There are three fundamental types of mutational events: (1) nucleotide deletions, (2) base-pair substitutions, and (3) gene duplications. Mutants that result from large deletions have the greatest stability. Mutants exhibiting high levels of reversion to wild-type cells are usually derived from base-pair substitution mutations. Alkylating agents are among the most potent direct-acting mutagenic agents.

A variety of methods has been developed for the production of mutants. The frequently used mutagen N-methyl N-nitrosoguanidine (nitrosoguanidine or NTG) functions by forming a methyl group adduct to guanine, and results in multiple mutations. Ethyl methane sulfonate is less lethal and usually results in single mutations. Although yielding large numbers of mutants, such agents usually result in mutations with significant rates of reversion to wild type. Exposure of cells to ultraviolet (UV) irradiation results in approximately equal numbers of both deletions and base-pair substitutions (16). UV irradiation will therefore yield a higher percent of more stable mutants (derived from frame shift mutations) and is the preferred method for mutagenesis. The usual procedure is to expose a cell suspension to UV irradiation to achieve a 50% reduction in viable cells and then to plate out the survivors to isolate mutants. This implies preliminary knowledge of the UV destruction rate. NTG, however, usually produces higher levels of mutagenesis than UV.

Overproducing riboflavin mutants of the mold Ashbya gossypii can initially be detected by visually observing the increased intensity of orange coloration of the colonies (17,18). Wild-type isolates of Pfaffia rhodozyma produce the red carotenoid pigment astaxanthan which imparts the characteristic pink coloration of salmon tissue, and they are initially a light pink. Sequential mutagenesis has been found to result in a seven- to tenfold increase in pigment production, which is readily detected by visually observing the colonies (19,20). The detection of amino acid overproducing colonies is greatly facilitated by plating overproducing cells (approximately 100 per plate) in the presence of large numbers of cells (approximately 108 ) of an appropriate auxotrophic indicator organism incapable of growth in the agar medium, used because of the absence of the amino acid to be overproduced. Overproduction by a colony is then visually observed and can be semiquan-titatively assessed on the basis of the diameter of the zone of growth of the indicator organism surrounding the amino acid overproducing colony.

To use Brevibacterium ammoniagenes to overproduce 5'-inosine monophosphate (5'-IMP) by direct fermentation, it is necessary to overcome feedback inhibition due to the synthesis of 5'-adenosine monophosphate (5'-AMP). This can be accomplished by muta-tionally blocking adenine synthesis while maintaining a low Mn++ level, which allows the membrane transit of 5'-IMP into the culture medium (21). The isolation of permeability mutants that allow secretion of 5'-IMP irrespective of Mn concentration has been a significant advance (22). The addition of low levels of adenine to the culture medium for growth is required.

Vegetarian Food and Cooking

Vegetarian Food and Cooking

Switch To A Vegetarian Diet And Live To Be A 100. Are You Suffering From Weak Bones And Digestive Disorders? Did You Ever Wonder About Why You Feel Restless, Listless Or Anxious Without Any Plausible Reason? The Answer Probably Lies In Your Dietary Habits.

Get My Free Ebook


Post a comment