Isolation methods of high glycosidase-producing mutants of Aspergillus luchuensis and its mutated genes.

High glycosidase-producing strains of Aspergillus luchuensis were isolated from 2-deoxyglucose (2-DG) resistant mutants. α-Amylase, exo-α-1,4-glucosidase, ß-glucosidase and ß-xylosidase activity in the mutants was ~3, ~2, ~4 and ~2.5 times higher than the parental strain RIB2604 on koji-making conditions, respectively. Citric acid production and mycelia growth of the mutants, however, approximately halved to that of the parent. Compared to the parent, the alcohol yield from rice and sweet potato shochu mash of the mutant increased ~5.7% and 3.0%, respectively. The mutant strains showed significantly low glucose assimilability despite the fructose one was almost normal, and they had a single missense or nonsense mutation in the glucokinase gene glkA. The recombinant strain that was introduced at one of the mutations, glkA Q300K, demonstrated similar but not identical phenotypes to the mutant strain. This result indicates that glkA Q300K is one of the major mutations in 2-DG resistant strains.

Histone deacetylases in sake yeast affect fermentation characteristics.

Yeast histone deacetylases (HDAC) affect the production of alcoholic beverages. In this study, we evaluated the sake fermentation characteristics when using HDAC gene-disrupted yeast strain Kyokai No. 701. Flavor components of the sake product were significantly changed. RPD3 or HDA1 disruption increased twofold the amount of isoamyl acetate, and isoamyl alcohol levels also increased in the rpd3Δ strain. To determine the contribution of Rpd3L and Rpd3S complexes to sake characteristics, a gene responsible for Rpd3L and/or Rpd3S formation was also disrupted. Disruption of DEP1 or SDS3 that is an essential component of Rpd3L led to increased isoamyl alcohol production similar to that of the rpd3Δ strain, but the efficiency of isoamyl alcohol esterification was not affected. In addition, Rpd3 and Hda1 may regulate the responsiveness to oxygen in isoamyl acetate production. We conclude that HDAC genes regulate the production of flavor components during sake fermentation. Abbreviations: HDAC: Histone deacetylase; HAT: histone acetyltransferase; K701: sake yeast Kyokai No. 701; PCR: polymerase chain reaction; HPLC: high performance liquid chromatography; E/A: Ester/Alcohol; BCAA: branched chain-amino acid; Atf: alcohol acetyltransferase.

Development of N- and O-linked oligosaccharide engineered Saccharomyces cerevisiae strain.

Yeast cells have been engineered for the production of glycoproteins as biopharmaceuticals with humanized N-linked oligosaccharides. The suppression of yeast-specific O-mannosylation is important to reduce immune response and to improve heterologous protein productivity in the production of biopharmaceuticals. However, so far, there are few reports of the engineering of both N-linked and O-linked oligosaccharides in yeast cells. In the present study, we describe the generation of a Saccharomyces cerevisiae strain capable of producing a glycoprotein with humanized Man5GlcNAc2 N-linked oligosaccharides, an intermediate of mammalian hybrid- and complex-type oligosaccharides, while suppressing O-mannosylation. First, a yeast strain that produces a glycoprotein with Man5GlcNAc2 was isolated by introducing msdS encoding α-1,2-mannosidase into a strain synthesizing Man8GlcNAc2 N-linked oligosaccharides. Next, to suppress O-mannosylation, an O-mannosyltransferase-deficient strain was generated by disrupting PMT1 and PMT2 Although the relative amount of O-linked oligosaccharides in the disruptant was reduced to approximately 40% of that in wild type cells, this strain exhibited growth defects and decreased protein productivity. To overcome the growth defects, we applied a mutagenesis technique that is based on the disparity theory of evolution. Finally, to improve protein productivity of the growth-recovered strain, vacuolar proteases PEP4 and PRB1 were further disrupted. Thus, by combining genetic engineering and disparity mutagenesis, we generated an Saccharomyces cerevisiae strain whose N- and O-linked oligosaccharide synthetic pathways were engineered to effectively produce the heterologous protein.

Protease-deficient Saccharomyces cerevisiae strains for the synthesis of human-compatible glycoproteins.

Saccharomyces cerevisiae strains engineered previously to produce proteins with mammalian high mannose structures showed severe growth defects and decreased protein productivity. In strain YAB101, derived from one of these strains by a mutagenesis technique based on the disparity theory of evolution, these undesirable phenotypes were alleviated. Here we describe further engineering of YAB101 with the aim of synthesizing heterologous glycoproteins with Man5GlcNAc2, an intermediate for the mammalian hybrid and complex type oligosaccharides. About 60% conversion of Man8GlcNAc2 to Man5GlcNAc2 was observed after integration of Aspergillus saitoi α-1,2-mannosidase fused to the transmembrane domain of S. cerevisiae Och1. To obtain a higher yield of the target protein, a protease-deficient version of this strain was generated by disruption of PEP4 and PRB1, resulting in YAB101-4. Inactivation of these vacuolar proteases enhanced the secretion of human interferon-ß by approximately 10-fold.

Construction of a long hairpin RNA expression library using Cre recombinase.

A large number of genome-wide screens using RNA interference (RNAi) libraries have been utilized to determine the function of individual gene products involved in a variety of biological processes. In this study, we describe a new method to enzymatically generate a long hairpin RNA (lhRNA) expression library from a cDNA plasmid library using a nicking endonuclease, BcaBEST DNA polymerase, and Cre recombinase without excising the inserted DNA fragment from the plasmid vector. This method involves 5 steps: (1) conversion of an inserted DNA fragment in a plasmid into a direct repeat (DR); (2) purification of the plasmid containing the DR; (3) subcloning a lox71 cassette into the plasmid; (4) conversion of the DR in the plasmid into an inverted repeat (IR) using Cre recombinase; and (5) purification of the plasmid containing the IR. We also established an efficient method for inserting DNase I-digested DNA fragments into expression plasmids to enable construction of a cDNA plasmid library suitable as source materials to construct the lhRNA expression library. We confirmed that each of the lhRNA expression plasmids constructed using this method induced strong RNAi in a silkworm cell line, NIAS-Bm-oyanagi2.

Inhibition assay of yeast cell walls by plasmon resonance Rayleigh scattering and surface-enhanced Raman scattering imaging.

We report on plasmon resonance Rayleigh scattering (PRRS) and surface enhanced Raman scattering (SERS) imaging for inhibition assay of yeast cell walls. This assay reveals that the proteins having alkali sensitive linkage bound to ß1,3 glucan frameworks in cell walls are involved in SERS activity. The result is further confirmed by comparison of genetically modified cells and wild type cells. Finally, we find that PRRS and SERS spots do not appear on cell walls when daughter cells are enough smaller than parent ones, but appear when size of daughter cells are comparable to parent cells. This finding indicates the relationship between expression of the proteins that generate SERS spots and cell division. These results demonstrate that PRRS and SERS imaging can be a convenient and sensitive method for analysis of cell walls.

A novel method to convert a DNA fragment inserted into a plasmid to an inverted repeat structure.

Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.

DNA vector-based RNA interference in cell lines derived from Bombyx mori.

Gene-knockdown technology using RNA interference (RNAi) is widely used to characterize gene functions in many organisms. In this study, we analyzed the conditions for employing DNA vector-based RNAi in silkworm cell lines using long-hairpin RNA-expressing plasmid DNA. We found that NIAS-Bm-oyanagi2 was the most effective cell line for RNAi. Expression of long-hairpin RNA containing an approximately 500 base-pair stem region suppressed expression of a reporter target gene by more than 99% in this cell line. Furthermore, the loop sequence of hairpin RNA was not as important to RNAi efficiency as previously observed in Drosophila melanogaster. DNA vector-based RNAi also induced significant suppression of endogenous clathrin in NIAS-Bm-oyanagi2. Luciferase activity from recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) containing luciferase in the clathrin-knockdown cells was significantly less than in the control cells, suggesting that clathrin is indispensable for the entry of BmNPV into silkworm cell lines.

shRNA expression plasmids generated by a novel method efficiently induce gene-specific knockdown in a silkworm cell line.

RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.

A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori.

A genome-wide analysis of innate immunity-related genes and gene families was conducted using the silkworm, Bombyx mori. We identified orthologs for a large number of genes involved in insect immunity that have been reported from Drosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera). B. mori has a unique recognition gene and antimicrobial peptide genes that are not present in the Drosophila, Anopheles, Apis and Tribolium genomes, suggesting a lineage-specific gene evolution for lepidopteran insects. The comparative analysis of the insect immune repertoires indicated a dynamic and flexible gene expansion in recognition, modulation and effector mechanisms due to different selection pressures. Differential gene regulation by different bacterial species was found in PGRP and Serpin genes, suggesting that Bombyx has a highly selective gene regulation system depending on bacterial species.

Binding of Bacillus thuringiensis Cry1A toxins to brush border membrane vesicles of midgut from Cry1Ac susceptible and resistant Plutella xylostella.

Plutella xylostella strain resistant (PXR) to Bacillus thuringiensis Cry1Ac toxin was not killed at even more than 1000 microg Cry1Ac/g diet but killed by Cry1Ab at 0.5 microg/g diet. In contrast, susceptible strain (PXS) was killed by Cry1Ac at 1 microg/g diet. Cy3-labeld Cry1A(s) binding to brush border membrane vesicles (BBMV) prepared from both strains were analyzed with direct binding assay. The Kd value of Cry1Aa to both BBMV was almost identical: 213.2 and 205.8 nM, and 263.5 and 265.0 nM for Cry1Ac. The highest Kd values were in Cry1Ab which showed most effective insecticidal activity in PXS and PXR, 2126 and 2463 nM, respectively. These results clearly showed that the BBMV from PXR and PXS could equally bind to Cry1Ac. The binding between BBMV and Cy3-labeled Cry1Ac was inhibited only by anti-175 kDa cadherin-like protein (CadLP) and -252 kDa protein antisera, but not by anti-120 kDa aminopeptidase. This supports that resistance in PXR resulted from the abortion of pore formation after the binding of Cry1Ac to the BBMV. And furthermore, the importance of 175K CadLP and P252 proteins in those bindings was suggested. We briefly discuss possible mechanisms of the resistance.

Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane.

Bacillus thuringiensis insecticidal proteins, Cry toxins, following ingestion by insect larvae, induce insecticidal effect by penetrating the brush border membranes (BBM) of midgut epithelial cells. Purified, activated B. thuringiensis Cry1Aa bound to Bombyx mori BBMV or unbound Cry1Aa were vigorously digested with Pronase. Both digests were compared by Western blotting. Free Cry1Aa was digested to alpha-helix and/or to amino acids at 1 mg Pronase/mL within 2.4 h at 37 degrees C. Whereas, BBMV-bound Cry1Aa was very resistant to Pronase digestion and even at 2 mg for 24 h, 7.5 kDa and approximately 30 kDa peptide were detected by alpha-2,3 antiserum, and alpha-4,5 and alpha-6,7 antisera, respectively. Another approximately 30 kDa peptide was also detected by beta-6-11 and domain III antisera. These fragments are believed either to be embedded in or to strongly interact with the BBMV. The 7.5 and former approximately 30 kDa peptides are thought to be derived from alpha-2,3 helix and stretch of alpha-4 to alpha-7 helices. Furthermore the latter approximately 30 kDa was thought to include the stretch of beta-6 to domain III. Moreover, the embedded Cry1Aa molecule appears to be segregated in some areas of beta-1-5 sheets, resulting in the above two approximately 30 kDa peptides. From these digestion patterns, we proposed new membrane insertion model for single Cry1Aa molecule. On the other hand, in digestion of BBMV-bound Cry1Aa, 15 kDa peptide which was recognized only by alpha-4,5 antiserum was observed. This fragment must be dimeric alpha-4,5 helices and we discussed the origin of this peptide.