Encapsulation enhances protoplast fusant stability

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.     

Human enteropeptidase light chain: Bioengineering of recombinants and kinetic investigations of structure and function

The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK～ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (k_cat/K_M = 6.83 × 10⁶ M^?1 sec^?1) and DDDDR (k_cat/K_M = 1.89 × 10⁷ M^?1 sec^?1) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge–charge interactions in the extended substrate binding pocket.     

Métabolisme de l’Acide 2-Aminoéthylphosphonique (Ciliatine) chez le Poulet

For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.     

Structural and Functional Properties of Chicken Lysozyme Fused Serine-rich Heptapeptides at the C-Terminus

Two serine-rich heptapeptides, Ser-Ser-Ser-Lys-Ser-Ser-Ser (S6K) and Ser-Ser-Ser-Ser-Ser-Ser-Ser (S7), were fused to the C-terminus of chicken lysozyme (Lz) by genetic modification to improve the functional properties of lysozyme. The cDNAs of S6K-lysozyme (S6K-Lz) and S7-lysozyme (S7-Lz) were inserted into the expression vector of Pichia pastoris and secreted in yeast cultivation medium. The secretion amounts of S6K-Lz and S7-Lz were about 60% of that of wild-type lysozyme (Wt-Lz). The CD spectra showed that the conformation of S6K-Lz and S7-Lz was conserved regardless of the attachment of serine-rich peptides. The denaturation curves of S6K-Lz and S7-Lz also showed that the conformational changes were very small. The lytic activity of S6K-Lz and S7-Lz was almost the same as that of Wt-Lz, while the bactericidal activity against Escherichia coli of S6K-Lz and S7-Lz was greatly increased. The acetic acid-urea PAGE of phosphatase-treated S6K-Lz and S7-Lz indicated the possibility of phosphorylation of the fused serine-rich heptapeptides.     

High-Level Expression of Fusion Protein Containing 10 Tandem Repeated GLP-1 Analogs in Yeast Pichia pastoris and Its Biological Activity in a Diabetic Rat Model

Glucagon-like peptide-1 (GLP-1), an incretin secreted by intestinal L-cells, can effectively lower blood glucose levels in patients with diabetes. A fusion gene, consisting of 10 tandem repeated GLP-1 analog genes, was expressed at a high level in the yeast Pichia pastoris. SDS polyacrylamide gel electrophoresis (SDS–PAGE), and Western Blotting results showed that fusion protein migrated as a single protein band with a molecular weight of 36 kDa. A biological activity test showed that the GLP-1 analog could significantly lower the level of serum glucose when GLP-1 purified analog was injected into diabetic rats. A potential strategy for large-scale production of fusion protein containing the 10 GLP-1 analogs as discovered, and a single GLP-1 analog was obtained from fusion protein digested with trypsin. This should be inspired foreign expression of medicinal short peptides and be valuable in further research on GLP-1 analog drugs in the treatment of diabetes mellitus.     

Gene Cloning of Cellobiohydrolase II from the White Rot Fungus Irpex lacteus MC-2 and Its Expression in Pichia pastoris

A gene (cel4) coding for a cellobiohydrolase II (Ex-4) was isolated from the white rot basidiomycete, Irpex lacteus strain MC-2. The cel4 ORF was composed of 452 amino acid residues and was interrupted by eight introns. Its deduced amino acid sequence revealed a multi domain structure composed of a cellulose-binding domain, a linker, and a catalytic domain belonging to family 6 of glycosyl hydrolases, from the N-terminus. cel4 cDNA was successfully expressed in the yeast Pichia pastoris. Recombinant Ex-4 showed endo-processive degrading activity towards cellulosic substrates, and a synergistic effect in the degradation of Avicel was observed when the enzyme acted together with either cellobiohydrolase I (Ex-1) or endoglucanase (En-1) produced by I. lacteus MC-2.     

Isolation of Marine Yeasts Collected from the Pacific Ocean Showing a High Production of γ-Aminobutyric Acid

Marine yeasts were collected from coastal and deep sea areas in the Pacific Ocean and the Sea of Japan around central and northern Japan to prepare a novel type of natural seasoning. It was found that one of the marine yeasts collected from the Pacific Ocean off Hachinohe showed a high concentration of γ-aminobutyric acid (GABA) in its extract, about 7–10 times higher than those of commercially available bread yeast and other marine yeasts. The marine yeast isolated and named Hachinohe No. 6 catalyzed the reaction from monosodium glutamate to GABA only in the presence of glucose. Subsequently, several marine yeasts belonging to the genera Pichia and Candida were found to have such catalytic activities, but not those belonging to the genus Saccharomyces. Isolate Hachinohe No. 6 was found to have the highest catalytic activity among the yeasts examined in this study.     

Production and Characterization of Recombinant Phanerochaete chrysosporium Cellobiose Dehydrogenase in the Methylotrophic Yeast Pichia pastoris

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.     

Purification and Characterization of Lipid Bioflocculant Produced by Rhodococcus erythropolis

The bioflocculant produced by Rhodococcus erythropolis S-1 was found to exist as huge assemblies, the molecular mass of which is over one million daltons, composed of many polypeptides and lipids in aqueous solution. We have isolated and purified this lipid bioflocculant by ultracentrifugation, extracting with 90% acetone, and two successive silica gel chromatographies from the culture broth. It was homogeneous on silica gel thin-layer chromatography. ¹H-NMR and HPLC studies showed that it was a kind of glycolipid that contained a C₁₆ methylene chain on the average and glucose in its chemical structure. The flocculating activity against kaolin clay suspension was dependent on the Ca²⁺ concentration.     

Production of Higher-quality Plastein from a Crude Single-cell Protein

From defatted n-paraffin-assimilating yeast cells, a crude protein was obtained by alkaliextraction followed by acid-precipitation. Then the protein was treated with ether until extractable substances were removed exhaustively at this stage. However, at the next stage where the ether-treated protein had been partially hydrolyzed with pepsin, when the hydrolysate was retreated with ether, it was found that ether-extractable substances totalling 270 mg/100 g were obtainable additionally. Chromatographic investigations demonstrated that the substances included significant amounts of aliphatic and aromatic hydrocarbons, some indoles, and a ubiquinone (n = 8).

From the protein hydrolysate (substrate) after the above ether-treatment, a plastein was synthesized with Bioprase under the specific conditions. The plastein was obtained as a precipitate when the whole reaction mixture was treated with aqueous ethanol or acetone. The quantity and quality (nitrogen content) of the plastein depended on the ethanol or acetone concentration. Roughly speaking, the higher the concentration, the more the plastein quantity. The converse relation held for the quality; a plastein precipitated by treatment solely with water showed a higher quality than any other case.