Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101.

In the present study, a novel oleaginous Thraustochytrid containing a high content of docosahexaenoic acid (DHA) was isolated from a mangrove ecosystem in Malaysia. The strain identified as an Aurantiochytrium sp. by 18S rRNA sequencing and named KRS101 used various carbon and nitrogen sources, indicating metabolic versatility. Optimal culture conditions, thus maximizing cell growth, and high levels of lipid and DHA production, were attained using glucose (60 g l⁻¹) as carbon source, corn steep solid (10 g l⁻¹) as nitrogen source, and sea salt (15 g l⁻¹). The highest biomass, lipid, and DHA production of KRS101 upon fed-batch fermentation were 50.2 g l⁻¹ (16.7 g l⁻¹ day⁻¹), 21.8 g l⁻¹ (44% DCW), and 8.8 g l⁻¹ (40% TFA), respectively. Similar values were obtained when a cheap substrate like molasses, rather than glucose, was used as the carbon source (DCW of 52.44 g l⁻¹, lipid and DHA levels of 20.2 and 8.83 g l⁻¹, respectively), indicating that production of microbial oils containing high levels of DHA can be produced economically when the novel strain is used.

Stimulation of reductive glycerol metabolism by overexpression of an aldehyde dehydrogenase in a recombinant Klebsiella pneumoniae strain defective in the oxidative pathway.

Previously, we constructed a glycerol oxidative pathway-deficient mutant strain of Klebsiella pneumoniae by inactivation of glycerol dehydrogenase (dhaD) to eliminate by-product synthesis during production of 1,3-propanediol (1,3-PD) from glycerol. Although by-product formation was successfully blocked in the resultant strain, the yield of 1,3-PD was not enhanced, probably because dhaD disruption resulted in insufficient regeneration of the cofactor NADH essential for the activity of 1,3-PD oxidoreductase (DhaT). To improve cofactor regeneration, in the present study we overexpressed an NAD(+)-dependent aldehyde dehydrogenase in the recombinant strain. To this end, an aldehyde dehydrogenase AldHk homologous to E. coli AldH but with NAD(+)-dependent propionaldehyde dehydrogenase activity was identified in K. pneumoniae. Functional analysis revealed that the substrate specificity of AldHk embraced various aldehydes including propionaldehyde, and that NAD(+) was preferred over NADP(+) as a cofactor. Overexpression of AldHk in the glycerol oxidative pathway-deficient mutant AK/pVOTHk resulted in a 3.6-fold increase (0.57 g l(-1) to 2.07 g l(-1)) in the production of 3-hydroxypropionic acid (3-HP), and a 1.1-fold enhancement (8.43 g l(-1) to 9.65 g l(-1)) of 1,3-PD synthesis, when glycerol was provided as the carbon source, compared to the levels synthesized by the control strain (AK/pVOT). Batch fermentation using AK/pVOTHk showed a significant increase (to 70%, w/w) in conversion of glycerol to the reductive metabolites, 1,3-PD and 3-HP, with no production of by-products except acetate.

Isolation and characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp. BR-2.

Forty-four eicosapentaenoic acid (EPA)-producing microbial strains were isolated from the intestines of marine fishes. Among them, one strain showing a maximum level of EPA (4.78%of total fatty acids) was identified as Shewanella sp. BR-2 on the basis of its 16S rRNA sequence. The EPA content reached a maximum level during the mid-exponential phase of cell growth, and gradually decreased with further growth of the cells. A cosmid DNA including the EPA biosynthesis gene cluster consisting of pfaA-E was isolated from a cosmid library of genomic DNA of Shewanella sp. BR-2, named pCosEPA-BR2. An E. coli clone harboring pCosEPA-BR2 produced EPA at a maximum level of 7.5%of total fatty acids, confirming the EPA biosynthesis activity of the cloned gene cluster.

Improved ethanol tolerance in Escherichia coli by changing the cellular fatty acids composition through genetic manipulation.

To investigate the effect of cellular fatty acids composition on ethanol tolerance in Escherichia coli, we overexpressed either des, encoding fatty acid desaturase from Bacillus subtilis, or fabA, encoding beta-hydroxydecanoyl thio-ester dehydrase from E. coli, or both genes together, into E. coli. Recombinant E. coli harboring fabA had elevated tolerance against ethanol compared to wild type strain. In contrast, des decreased resistance to ethanol. Co-expression of both genes together complemented ethanol tolerance of E. coli. This result indicates how to engineer bacterial strains to be resistant to higher concentrations of ethanol.

Elimination of by-product formation during production of 1,3-propanediol in Klebsiella pneumoniae by inactivation of glycerol oxidative pathway.

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae involves the formation of various by-products, which are synthesized through the oxidative pathway. To eliminate the by-products synthesis, the oxidative branch of glycerol metabolism was inactivated by constructing two mutant strains. In one of the mutant strains, the structural genes encoding glycerol dehydrogenase and dihydroxyacetone kinase were deleted from the chromosomal DNA, whereas in the second mutant strain dhaR, which is a putative transcription factor that activates, gene expression was deleted from the chromosomal DNA. In the resultant mutant strains lacking the dhaT gene encoding 1,3-PD oxidoreductase, which was simultaneously deleted while replacing the native promoter with the lacZ promoter, the by-product formation except for acetate was eliminated, but it still produced 1,3-PD at a lower level, which might be due to a putative oxidoreductase that catalyzes the production of 1,3-PD. The recombinant strains in which the reductive pathway was recovered produced slightly lower amount of 1,3-PD as compared to the parent strain, which might be due to the reduced activity of DhaB caused by the substitution of the promoter. However, the production yield was higher in the recombinant strain (0.57 mol mol(-1)) than the wild type Cu strain (0.47 mol mol(-1)).

An efficient plasmid vector for constitutive high-level expression of foreign genes in Escherichia coli.

The levansucrase gene (lsrA) from Rahnella aquatilis was strongly expressed in a constitutive manner in Escherichia coli when cloned into a pBluescript KS-based pRL1CP plasmid vector. The native promoter upstream of lsrA and the lacZ promoter cooperatively enhanced the expression of lsrA to a level that was comparable to that of the T7 promoter, which is used in commercial pET expression vector system. A putative rho-independent transcription termination signal downstream of lsrA was crucial for gene expression. This plasmid vector also proved to be applicable for efficient expression of other foreign genes in E. coli.

Identification of erythrocyte p55/MPP1 as a binding partner of NF2 tumor suppressor protein/Merlin.

Neurofibromatosis type 2 is an inherited disorder characterized by the development of benign and malignant tumors on the auditory nerves and central nervous system with symptoms including hearing loss, poor balance, skin lesions, and cataracts. Here, we report a novel protein-protein interaction between NF2 protein (merlin or schwannomin) and erythrocyte p55, also designated as MPP1. The p55 is a conserved scaffolding protein with postulated functions in cell shape, hair cell development, and neural patterning of the retina. The FERM domain of NF2 protein binds directly to p55, and surface plasmon resonance analysis indicates a specific interaction with a kD value of 3.7 nM. We developed a specific monoclonal antibody against human erythrocyte p55, and found that both p55 and NF2 proteins are colocalized in the non-myelin-forming Schwann cells. This finding suggests that the p55-NF2 protein interaction may play a functional role in the regulation of apico-basal polarity and tumor suppression pathways in non-erythroid cells.

Alternatively spliced exon 5 of the FERM domain of protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells.

Direct physical linkage of MAGUKs to the actin cytoskeleton was first established by the interaction of erythrocyte p55 with the FERM domain of protein 4.1R. Subsequently, it was reported that p55 binds to a 51-amino acid peptide, encoded by exon 10, located within the FERM domain of protein 4.1R. In this study, we investigated the nature of the p55-FERM domain binding interface and show that p55 binds to a second 35-amino acid peptide, encoded by an alternatively spliced exon 5, located within the FERM domain of protein 4.1R. Competition and Surface Plasmon Resonance-binding measurements suggest that the peptides encoded by exons 5 and 10 bind to independent sites within the D5 domain of p55. Interestingly, the full length 135 kDa isoform of protein 4.1R containing both exons 5 and 10 was targeted exclusively to the plasma membrane of epithelial cells whereas the same isoform without exon 5 completely lost its membrane localization capacity. Together, these results indicate that p55 binds to two distinct sites within the FERM domain, and the alternatively spliced exon 5 is necessary for the membrane targeting of protein 4.1R in epithelial cells. Since sequences similar to the exon 5-peptide of protein 4.1R and D5 domain of p55 are conserved in many proteins, our findings suggest that a similar mechanism may govern the membrane targeting of other FERM domain containing proteins.

Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

Purification of the NF2 tumor suppressor protein from human erythrocytes.

BACKGROUND: Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane. METHODS: Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a approximately 70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins. RESULTS: These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately 41-65,000 molecules of NF2 protein per erythrocyte. CONCLUSION: We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.

Cloning and characterization of Plasmodium falciparum cysteine protease, falcipain-2B.

The gene for malaria parasite cysteine protease falcipain-2B has been isolated from the Plasmodium falciparum genomic DNA. Falcipain-2B gene is located adjacent to the falcipain-2A gene on chromosome 11, and the two enzymes show extensive sequence identity at the amino acid level. Using reverse transcribed polymerase chain reaction (RT-PCR), the transcript of falcipain-2B was detected at the trophozoite stage of P. falciparum in human erythrocytes. Recombinant falcipain-2B protein expressed in bacteria exhibits protease activity as established by the cleavage of fluorescent peptide substrate as well as in-gel gelatin zymography. Importantly, the recombinant falcipain-2B cleaved host ankyrin but not protein 4.1 as assessed by the erythrocyte inside-out-vesicle assay in vitro. Notwithstanding its predicted hemoglobinase function, the P. falciparum falcipain-2B may contribute and orchestrate selective proteolytic events during the exit of malaria parasite from human red blood cells.

The phylogenetic relationships of cyanobacteria inferred from 16S rRNA, gyrB, rpoC1 and rpoD1 gene sequences.

Phylogenetic analysis of cyanobacteria was carried out using the small subunit rRNA (16S rRNA), DNA gyrase subunit B (gyrB), DNA-dependent RNA polymerase gamma subunit (rpoC1) and a principal sigma factor of E. coli sigma(70) type for DNA-dependent RNA polymerase (rpoD1) gene sequences of 24 strains which contained 5 subgroups of cyanobacteria-3 strains of the Chroococcales, 5 strains of the Pluerocapsales, 7 strains of the Oscillatoriales, 7 strains of the Nostocales and 2 strains of the Stigonematales. Degenerated PCR primers of gyrB, rpoC1 and rpoD1 genes were designed using consensus amino acid sequences registered in GenBank. The phylogenetic positions of cyanobacteria were resolved through phylogenetic analysis based on 16S rDNA, gyrB, rpoC1 and rpoD1 gene sequences. Phylogenies of gyrB, rpoC1 and rpoD1 support 16S rRNA-based classification of cyanobacteria. Interestingly, phylogenies from amino acid sequences deduced from gyrB and combined amino acid sequences deduced from rpoC1 and rpoD1 genes strongly support that of 16S rRNA, but the branching pattens of the trees based on 16S rDNA, GyrB, rpoC1, rpoD1 and combined amino acid sequences deduced from rpoC1 and rpoD1 were not congruent. In this study, we showed the correlation among phylogenetic relationships of 16S rDNA, gyrB, rpoC1 and rpoD1 genes. The phylogenetic trees based on the sequences of 16S rDNA, GyrB, rpoC1, rpoD1 and the combined amino acid sequences deduced from rpoC1 and rpoD1 showed that the lateral gene transfer of rRNA might be suspected for Synechocystis sp. PCC 6803.