4.1.99.12 A137T naturally occuring Rib3 mutant which is partially deficient in cytochromes a, a3, and cytochrome b, the respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1. growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin, 2. the levels of flavin nucleotides are not significantly different in the mutant and wild type, phenotype, overview 674407 4.1.99.12 C59A site-directed mutagenesis, the mutant shows 70% of wild-type enzyme activity 675342 4.1.99.12 D21E site-directed mutagenesis, inactive mutant 674406 4.1.99.12 D21N site-directed mutagenesis, inactive mutant 674406 4.1.99.12 D30E site-directed mutagenesis, inactive mutant 674406 4.1.99.12 D32A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme -, 728745 4.1.99.12 D92A site-directed mutagenesis, inactive mutant 675342 4.1.99.12 E154D DBPS activity is abolished 716533 4.1.99.12 E163A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme -, 728745 4.1.99.12 E166A site-directed mutagenesis, inactive mutant 675342 4.1.99.12 E185D site-directed mutagenesis, inactive mutant 674406 4.1.99.12 E185X site-directed mutagenesis, inactive mutant 674408 4.1.99.12 E26D site-directed mutagenesis, inactive mutant 674406 4.1.99.12 E26Q site-directed mutagenesis, inactive mutant 674406 4.1.99.12 E28D site-directed mutagenesis, inactive mutant 674406 4.1.99.12 E30A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme -, 728745 4.1.99.12 E39A site-directed mutagenesis, inactive mutant -, 748186 4.1.99.12 E41A site-directed mutagenesis, the mutant is almost inactive 748186 4.1.99.12 F139A site-directed mutagenesis, the mutant shows moderately reduced activity compared to the wild-type enzyme -, 748186 4.1.99.12 H125A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme -, 728745 4.1.99.12 H142A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme -, 728745 4.1.99.12 H147S site-directed mutagenesis, the mutant enzyme shows about 10% of the wild-type enzyme activity 674408 4.1.99.12 H147S site-directed mutagenesis, the mutant shows about 10% of wild-type enzyme activity, crystal structure determination with bound substrate 671038 4.1.99.12 H154A site-directed mutagenesis, inactive mutant -, 748186 4.1.99.12 H164N site-directed mutagenesis, inactive mutant 674406 4.1.99.12 M84A site-directed mutagenesis, the mutant shows moderately reduced activity compared to the wild-type enzyme 748186 4.1.99.12 additional information an additional single copy of the ribA gene introduced into the sacBlocus of the riboflavin production strain and constitutive expression from the medium strength vegI promoter leads to improved riboflavin titers and yields of riboflavin on glucose of up to 25%, strain VB2XL1, both enzymatic activities of RibA, the 3,4-dihydroxy-2-butanone 4-phosphate synthase activity located in the N-terminal half of the protein and the GTP cyclohydrolase II activity of the C-terminal domain, are necessary for the improved riboflavin productivity, method, overview 675226 4.1.99.12 additional information construction of RIB3 disruption mutants, restoration by riboflavin of growth of a rib3 deletion mutant on glucose but not glycerol/ethanol, phenotype, overview 674407 4.1.99.12 additional information engineering of Escherichia coli for increased riboflavin production: overexpression of gene ribB to increase the flux from ribulose 5-phosphate to 3,4-dihydroxybutan-2-one 4-phosphate. Then ndk and gmk genes are overexpressed to enhance GTP supply. Subsequently, a R419L mutation is introduced into purA to reduce the flux from IMP to AMP. Co-overexpression of mutant purF and prs genes further increased riboflavin production. The final strain RF18S produces 387.6 mg riboflavin per liter with a yield of 44.8 mg riboflavin per gram glucose in shake-flask fermentations. The final titer and yield are 72.2% and 55.6%. Mutant strain and method evaluation, overview -, 747595 4.1.99.12 N89A site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme -, 748186 4.1.99.12 Q181R/Q183R site-directed mutagenesis, construction of a synthetic gene, derived from orf 6.2440, with nucleotide exchanges at positions 414, 426, 477, 480, and 581 675342 4.1.99.12 R61A site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme 748186 4.1.99.12 S64A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme -, 748186 4.1.99.12 T143A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme 728745 4.1.99.12 T96A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme 728745 4.1.99.12 V98A site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme 748186 4.1.99.12 Y87A site-directed mutagenesis, the mutant shows 2% of wild-type enzyme activity 675342