Phylogenetic Analysis Of Iron-binding Proteins,Construction Of A Surface Display System And The Stress Tolerance Of Ethanolic Fermentation

Iron is an essential nutrient for all living organisms, playing important role in various biochemical process, such as electron transfer reactions, oxygen binding, and regulation of cell growth and differentiation. However, it is toxic of excess iron in the cellular, and free hydroxyl radicals can catalyze the oxidative damage of biomolecules. Living organisms can sequester and storage iron using high-affinity iron-binding molecules, which are ferritin family and transferrin family. The lactoferrin is an antimicrobial peptide of the transferrin family, which play roles in the immune and inflammatory responses. This study assessed the putative ferritin genes and transferrin genes in genomes from various species, and investigated the evolutionary scenario of these important iron-binding families, and the evolutionary relationship of lactoferrin with other members in transferrin family; we also use food-grade bacterium Lactococci lactis MG1614 for the expression and surface display of bovine-lactoferrin gene; meanwhile, this study focused on the performance of Lactococci lactis 12AE1 in high glucose concentration and various stresses corn stover hydrolysate. The main results as follows:1 The phylogenetic relationship of ferritin familyFerritin are highly conserved proteins that are widely distributed in various species from archaea to humans. This study investigated the ferritin genes in 248 genomes from viruses, archaea, bacteria, and eukarya. The distribution comparison suggests that mammals and eudicots possess abundant ferritin genes, whereas fungi contain very few ferritin genes. Archaea and bacteria show considerable numbers of ferritin genes. Generally, prokaryotes possess three types of ferritin(the typical ferritin, bacterioferritin, and DNA-binding protein from starved cell), whereas eukaryotes have various subunit types of ferritin, thereby indicating the individuation of the ferritin family during evolution. The characteristic motif analysis of ferritins suggested that all key residues specifying the unique structural motifs of ferritin are highly conserved across three domains of life. Meanwhile, the characteristic motifs were also distinguishable between ferritin groups, especially phytoferritins, which show a plant-specific motif. The phylogenetic analyses show that ferritins within the same subfamily or subunits are generally clustered together. The phylogenetic relationships among ferritin members suggest that both gene duplication and horizontal transfer contribute to the wide variety of ferritins, and their possible evolutionary scenario was also proposed. The results contribute to a better understanding of the distribution, characteristic motif, and evolutionary relationship of the ferritin family.2 The phylogenetic relationship of transferrin familyTransferrins have been identified in animals and green algae, and they consist of a family of evolutionarily related proteins that play a central role in iron transport, immunity, growth and differentiation. This study assessed the transferrin genes among 100 genomes from a wide range of animal and plant kingdoms. The results showed that putative transferrins were widespread in animals, but their gene quantity and type differ greatly between animal groups. Generally, Mammalia possess abundant transferrin genes, whereas Trematoda contain few ones. Melanotransferrin and serotransferrin are widely distributed in vertebrates, while melanotransferrin-like and transferrin-like 1 are frequent in invertebrates. However, only a few plant species detected putative transferrins, and a novel transferrin member was first uncovered in Angiospermae and Pteridophyta. The structural comparison among transferrin family members revealed seven very well-repeated and conserved characteristic motifs, despite a considerable variation in the overall sequences. The phylogenetic analysis suggested that gene duplication, gene loss and horizontal transfer contributed to the diversification of transferrin family members, and their inferred evolutionary scenario was proposed. These findings help to the understanding of transferrin distribution, characteristic motifs and residues, and evolutionary process.3 The expression and surface display of lactoferrin gene in food-grade bacterium Lactococci lactisA novel food-grade expression and surface location vector for lactococci lactis MG1614, pMG36-acmA-bLfcin-nis I,was developed using the Nisin-controlled gene(nisI) as a selection maker, and this novel vector was used for the expression and surface display of bovine-lactoferricin as a reporter gene; in addition, acmA as a functional anchoring gene was inserted for the efficient display heterologous peptides at the surface of food-grade bacterium L.lactis MG1614. The results show that the construction of food-grade expression and surface location vector in this research could be used for the industrial production of lactoferrin in lactococci lactis for the medicinal purpose and also for the production food materials.4 The tolerance of Lactobacillus casei 12AE1 in synthetic corn stover hydrolysate during fermentationDue to lactic acid bacteria commonly found in biofuel fermentations, we confirmed that lactic acid bacteria can utilize lignocellulosic hydrolysate-derived sugars, tolerate to inhibitors presents in lignocellulosic hydrosate and the pH change during fermentation. L.casei 12AE1 has been selected as the biofuels parental strain based upon its alcohol tolerance(>10% ethanol), carbohydrate utilization, and relatively high transformation efficiency. Hence, this study focused on the performance of three ethanologenic strains in high glucose concentration, acetate stress, and lignotoxins, in addition, the ethanol production of these strains to grow on synthetic corn stover hydrolysate was reported. The results show that L.casei 12AE1 can tolerate inhibitors better than other biocatalysts. The HPLC analysis show that L.casei 12AE1 has great growth performance and highest ethanol production. Our results suggest that all biocatalyst maintains high final cell densitied and growth rate in osmotic stress media and poor cell densitied in lignotoxins stress, possibly a direct osmoprotectant from amino acid. We also found that the metabolizm of Embden-Meyerhof-Parnas pathway was still worked, which converts pyruvate to lactate, and further study will be needed to improvement the carbohydrate utilization of L.casei 12AE1 in synthetic corn stover hydrolysate.