Construction Of Iron-enriched Yeast Bioengineering Strains And Studies On Biotransformation, Distribution, Combination State Of Iron In Cells Of Saccharomyces Cerevisiae

Iron is an essential nutrient for the human and animal. It is needed to form hemoglobin in blood and myoglobin in muscles. The deficiency of iron may result in an anaemia. Though iron is abundant in the environment, it is largely present as insoluble oxyhydroxides. Inorganic iron may not be as easily absorbed or biologically active as organic iron. Microelements from organic sources are more suitable and advantageous for both human and animal than those obtained from inorganic sources. Yeasts can accumulate many microelements including iron, and iron-enriched yeast could be used to prevent anaemia and related diseases. However, the storage capacities of iron in cells have large difference among different yeast strains. The main results are showed as follows:1. The production of iron-enriched yeast depends on not only iron content of cells, but also biomass. Generally, high biomass was accompanied by low iron content of cells or high iron content of cells was accompanied by low biomass in wild strains. A high-biomass, iron-enriched yeast fusant strain ZYF-15 was obtained by primary screening from different genera and species, second screening,isolation of haploid, DES mutagenesis and interspecies protoplasts fusion, which combined the superior properties of parental strains. The total iron content of the fusion strain was 1.7 and 1.3 times that of parental strains, respectively.2. The effects of different culture media, medium composition and cultivation conditions on the biomass and iron content of the strain ZYF-15 were tested with single factor experiments. The optimum medium contained (g/L): glucose 80, peptone 5, yeast extract 10. The biomass and iron content of strain ZYF-15 reached 11.2 g/L and 24.5 mg/g dry cells when cultured in the optimized medium as above with 600 ng/mL Fe2+, 50 mL/250mL volume, 10%(v/v) inoculum volume for 30 h at 30C. Initial pH value was adjusted to 7.0. Under these conditions, the biomass(dry weight) and iron content of strain ZYF-15 reached 11.2 g/L and 24.5 mg Fe/g dry cells. The total enriched iron content of cells was 1.5-fold more over of that of cells cultured under the initial conditions.A L9 34 orthogonal design experiment were performed for the strain ZYF-15 to measure the significance of factors. Glucose concentration was most vital factor to effect biomass of the strain ZYF-15, iron concentration was most important factor to effect iron content of cells of the strain, and peptone was most important factor to effect the total enriched iron content of the cells.In 5 L fermentor, the biomass and iron content of strain ZYF-15 reached 9.3 g/L and 39.8 mg/g dry cells when cultured in the condition described as above with 3 L/5 L volume, 20%(v/v) inoculum volume and pH 5= 4.5 during fermentation for 16 h at 30癈. The total enriched iron content of cells was 1.4-fold more over of that of cells cultured in shake flasks.3. Yeast cells are capable of biotransformation. The conversion of iron enriched from inorganic iron to organic iron was analyzed in the iron-enriched yeast cells. The fresh yeast cells were fractionated, and the distribution and combination state of iron was examined. The inorganic iron about 45.7% was incorporated into the yeast cells during the strain cultivating in the medium containing 600 ug/mL Fe2+, and about 97.6% of enriched iron was converted intoorganic iron, which is mainly in cell walls and vacuoles with some bound to DNA, RNA and protein.4. Flocculation of yeast cells is important to fermentation process, such as brewing and biological production of ethanol. The flocculation of yeast cells is capable to obsorb metal ion. So, the plasmid pIFC, an E.coli-yeast shuttle integration expression plasmid, contanining flocculation gene (FLO1G) and CUP1p-MT1 gene expression box was constructed. Plasmid pEF-1, containing a more copies flocculation gene and pIFC were introduced into iron-enriched yeast strain ZYF-15, and the flocculation of recombinant strains was assay. The effects of recombinant strains on the enriched iron content of cells was discussed. The...