| Non-Scientific Abstract Amino acids are widely produced and used as food
additives, animal feed supplements, cosmetics, and medicines. In many cases, bacteria are used to make
these amino acids. Isoleucine is one amino acid that relies on bacteria for its production. In an
effort to make the bacterial production of isoleucine more efficient, scientists are studying possible
enzymes that have an effect on isoleucine production.One of these genes that has recently been
suspected as being a part of the regulation of isoleucine is mmc. In our studies,we attempt to
learn about the promoter of mmc, so that its effect on isoleucine production can be better
determined.
Abstract
Corynebacterium glutamicum is used to produce amino acids such as isoleucine. Two different
threonine dehydratases from Escherichia coli have been used to create an isoleucine overproducing
strain.The less effective strain has been found to upregulate methylmalonyl CoA mutase (mmc) in late
stationary phase. In this project we attempted to examine the transcriptional regulatory mechanism of
mmc, as well as identify the necessary promoter sequence. PCR mutagenesis was used to create three
promoter deletions, which were tested by using transcriptional fusions to a reporter gene. It was
found that the necessary sequence of the mmc promoter begins somewhere between nucleotide 136 and 246,
upstream of the translational start site of the mmc gene. GUS assays were completed on the mmc
promoter fused to a promoterless reporter gene under many different conditions. These experiments
showed that mmc is not transcriptionally regulated by threonine, (-ketobutyrate, 3- hydroxybutyrate,
or vitamin B12 under the conditions tested. Isoleucine and 2-hydroxybutyrate appear to have some
effect on the timing of mmc transcription. |
