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Thymidine is an important precursor in antiviral drugs. We have enhanced thymidine production in E. coli by eliminating the repressors in the transcription of the gene coding for carbamoyl phosphate synthetase. The operon for carbamoyl phosphate synthetase (CarAB) in the thymidine biosynthesis regulatory pathway was derepressed by disrupting three known repressors (purR, pepA and argR). Combinatorial disruption of three repressors increased CarA expression levels in accordance with degree of disruption, which had a positive correlation with thymidine production. By simultaneous disruption of three repressors (BLdtugRPA), CarA expression level was increased by 3-fold compared to the parental strain, leading to an increased thymidine yield from 0.25 to 1.1 g thymidine l(-1). From BLdtugRPA, we established BLdtugRPA24 by transforming two plasmids expressing enzymes in the thymidine biosynthetic pathway and obtained 5.2 g thymidine l(-1) by Ph-stat fed-batch fermentation.

The most efficient substrate for mannitol production by Candida magnoliae HH-01 is fructose; glucose and sucrose can also be converted into mannitol but with lower conversion yields. Mannitol dehydrogenase was purified and characterized; it had the highest activity with fructose as the substrate and used only NADPH. In fed-batch fermentation with glucose, the production of mannitol from fructose ceased when the glucose was exhausted but it was reinitiated with the addition of glucose, implying that glucose plays an important role in NADPH regeneration.

Mutagenesis of Erwinia rhapontici was performed to enhance the production of isomaltulose from sucrose. A mutant strain, BN 68089, was obtained through a screening process involving automated and miniaturized cultivation in Bioscreen C. This high-throughput, miniaturized screening system was optimized to identify the mutant strain, which had a conversion yield (90%) and productivity (194 g l−1 h−1). The BN 68089 mutant cells were immobilized in sodium alginate and when operated in a packed bed reactor gave a yield of 89% and a productivity of 144 g l−1 h−1 of at 30 °C, the optimal temperature. Immobilized BN 68089 cells exhibited 8% and 15% higher yield and productivity, respectively, than those of the wild-type strain.

Supplementation with CaCl2⋅2H2O (40 mg l−1) or CuSO4⋅5H2O (10 mg l−1) improved vancomycin production by Amycolatopsis orientalis by 12 and 11%, respectively; used in combination, they acted synergistically. Ca2+ decreased the intracellular concentration of vancomycin 36%, whereas Cu2+ increased the intracellular activity of TDP-glucose:aglycosylvancomycin glucosyltransferase 3 times more than control. Ca2+ probably works by altering the permeability of cells to vancomycin, whereas Cu2+ increases the activity of an enzyme responsible for vancomycin biosynthesis.

A novel microorganism was isolated which is able to produce mannitol when grown in the presence of fructose and glucose as carbon sources. In flask culture in a medium containing 150 g fructose l−1, it yielded 67 g mannitol l−1 after 168 h. In fed-batch culture with 3–12% (w/v) fructose, production reached a maximum of 209 g mannitol l−1 after 200 h, corresponding to an 83% yield and a 1.03 g l−1 h−1 productivity. The isolated strain was identified as Candida magnoliae based on identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.

Of the vitamins tested, inositol was the most effective for erythritol production. To increase erythritol production by Torula sp., inositol and a related compound, phytic acid (myoinositol hexaphosphate), were added to the culture media. Erythritol production in the presence of phytic acid was greater than that in the presence of inositol, due to the synergistic effects of phosphate and inositol. Supplementation with phosphate and inositol increased cell growth, erythritol production, and the activity of erythrose reductase in cells. Inositol was a more effective stimulator of cell growth and erythritol production than was phosphate.

The production of erythritol and the erythritol yield from glucose by Torula sp. were improved, in increasing order, by supplementing with 10 mg MnSO4 ⋅ 4H2O l−1, 2 mg CuSO4 ⋅ 5H2O l−1, and both 10 mg MnSO4 ⋅ 4H2O l−1 and 2 mg CuSO4 ⋅ 5H2O l−1. Mn2+ decreased the intracellular concentration of erythritol, whereas Cu2+ increased the activity of erythrose reductase in cells. These results suggest that Mn2+ altered the permeability of cells, whereas Cu2+ increased the activity of erythrose reductase in cells.

Cell growth increased proportionally to the initial concentration of ammonium ion, however, methylan production was significantly inhibited at the high concentration of ammonium ion. The control of ammonium ion within the desired level(usually 0.45 g/l) was needed to reduce the inhibition. Methylan production was increased to 12.5 g/l by maintaining ammonium ion below 0.15 g/l.

Metal adsorption of the acidic polysaccharide produced from Methylobacterium organophilum was investigated. The polysaccharide non-specifically adsorbed 21% for copper and 18% for lead of the polysaccharide used after 30 minutes of reaction time at pH 7. The metal adsorption may be caused by the interaction between metal cations and negative charges of acidic residues of the polysaccharide such as uronic, pyruvic and acetic acid.

Intracellular NADPH/NADP+ ratio in cells grown on various production media with different carbon and nitrogen sources had a positive correlation with the thymidine production. To improve thymidine production in a previously engineered E. coli strain, NAD+ kinase was overexpressed in it resulting in the NADPH/NADP+ ratio shifting from 0.184 to 0.267. The [NADH + NADP+]/[NAD+ + NADPH] ratio was, however, not significantly altered. In jar fermentation, 740 mg thymidine l-1 was produced in parental strain, while 940 mg l-1 of thymidine was produced in NAD+ kinase-expressing strain.

Various emulsan samples with the different degrees of branching of the carbohydrate backbone were obtained from Acinetobacter calcoaceticus under different culture conditions. The emulsifying activity of emulsan had a linear correlation to the branching degrees of the carbohydrate backbone (r2= 0.930) suggesting that the structure of carbohydrate backbone was an important factor influencing emulsifying activity.

Specific erythritol production rate by Trigonopsis variabilis increased from 0.09 to 0.19 g/g-day by varying the osmotic pressure from 1.3 to 3.9 kPa in glucose medium, but the specific growth rate decreased from 0.28 to 0.13 h -1 . Therefore, osmotic pressure was adjusted to 1.4 kPa during growth phase and to 3.7 kPa during production phase in a two-stage fermentation. Erythritol reached 46 g/ l in such a system and was twice that obtained in one-stage fermentation.

Mutagenesis of Erwinia rhapontici was performed to enhance the production of isomaltulose from sucrose. A mutant strain, BN 68089, was obtained through a screening process involving automated and miniaturized cultivation in Bioscreen C. This high-throughput, miniaturized screening system was optimized to identify the mutant strain, which had a conversion yield (90%) and productivity (194 g l(-1) h(-1)). The BN 68089 mutant cells were immobilized in sodium alginate and when operated in a packed bed reactor gave a yield of 89% and a productivity of 144 g l(-1) h(-1) of at 30 degrees C, the optimal temperature. Immobilized BN 68089 cells exhibited 8% and 15% higher yield and productivity, respectively, than those of the wild-type strain.

The most efficient substrate for mannitol production by Candida magnoliae HH-01 is fructose; glucose and sucrose can also be converted into mannitol but with lower conversion yields. Mannitol dehydrogenase was purified and characterized; it had the highest activity with fructose as the substrate and used only NADPH. In fed-batch fermentation with glucose, the production of mannitol from fructose ceased when the glucose was exhausted but it was reinitiated with the addition of glucose, implying that glucose plays an important role in NADPH regeneration.