| |
| author |
Ryan Money
| | title |
Mutations of Bacillus subtilis to Increase Production of Riboflavin (Vitamin B2) Intermediate Guanosine Triphosphate (GTP)
| | abstract |
B. subtilis is a Gram-positive bacterium that produces a wide range of naturally occurring
products. A few of these products are antibiotics, such as sublancin or surfactin, and vitamins,
such as riboflavin (vitamin B2). Riboflavin is a natural vitamin that is an important part of the
function of the electron transport chain (ETC) within cells. Through this project, the production
of the key precursor to riboflavin, guanosine triphosphate (GTP), was increased by isolating
purine analog-resistant mutants of B. subtilis that redirect the carbon flow to guanine and away
from adenine. To do this, a library of randomly mutated B. subtilis was prepared using EMS
(ethyl methyl sulfonate) mutagenesis, and mutants were then screened for azaguanine (Az)
resistance, a toxic analog of guanine. Increasing guanine levels should correlate to an increase in
GTP levels. Azaguanine acts as a competitive inhibitor of biosynthetic enzymes that convert
guanine to GTP; the B. subtilis strain used in the EMS mutagenesis contains a preexisting purB
mutation that enhances the recovery of azaguanine-resistant mutations in the GMP-IMP-AMP
junction of the purine pathway. Twelve azaguanine-resistant mutants (Azr) were recovered and
characterized for azaguanine resistance using a maximum inhibitory concentration (MIC) assay,
and guanine levels were measured using a reverse halo (growth halo) plate assay and High-
Performance Liquid Chromatography (HPLC). Of these 12 mutants, one mutant, Az8, was
determined to have the highest-produced guanine; the next best was a spontaneous mutant, Az2.
We speculated that the mutation in Az8 favors the production of guanine and guanosine over the
production of hypoxanthine or inosine. In the future, the Az8 strain will be used to further
increase guanine/GTP production in a second mutagenesis and selection round, using the
guanosine toxic analog decoyinine.
| | school |
The College of Liberal Arts, Drew University
| | degree |
B.S. (2023)
|
| advisor |
Dr. John Perkins
|
| full text | RMoney.pdf |
| |
