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Light stimulates carotenoid production in an oleaginous yeast Rhodosporidium toruloides NBRC 10032 by promoting carotenoid biosynthesis genes. These genes undergo two-step transcriptional activation. The potential light regulator, Cryptochrome DASH (CRY1), has been suggested to contribute to this mechanism. In this study, based on KU70 (a component of nonhomologous end joining (NHEJ)) disrupting background, CRY1 disruptant was constructed to clarify CRY1 function. From analysis of CRY1 disruptant, it was suggested that CRY1 has the activation role of the carotenogenic gene expression. To obtain further insights into the light response, mutants varying carotenoid production were generated. Through analysis of mutants, the existence of the control two-step gene activation was proposed. In addition, our data analysis showed the strong possibility that R. toruloides NBRC 10032 is a homo-diploid strain.

The oleaginous yeast Lipomyces starkeyi is an intriguing lipid producer that can produce triacylglycerol (TAG), a feedstock for biodiesel production. We previously reported that the L. starkeyi mutant E15 with high levels of TAG production compared with the wild-type was efficiently obtained using Percoll density gradient centrifugation. However, considering its use for biodiesel production, it is necessary to further improve the lipid productivity of the mutant. In this study, we aimed to obtain mutants with better lipid productivity than E15, evaluate its lipid productivity, and analyze lipid synthesis-related gene expression in the wild-type and mutant strains. The mutants E15-11, E15-15, and E15-25 exhibiting higher lipid productivity than E15 were efficiently isolated from cells exposed to ultraviolet light using Percoll density gradient centrifugation. They exhibited approximately 4.5-fold higher lipid productivity than the wild-type on day 3. The obtained mutants did not exhibit significantly different fatty acid profiles than the wild-type and E15 mutant strains. E15-11, E15-15, and E15-25 exhibited higher expression of acyl-CoA synthesis- and Kennedy pathway-related genes than the wild-type and E15 mutant strains. Activation of the pentose phosphate pathway, which supplies NADPH, was also observed. These results suggested that the increased expression of acyl-CoA synthesis- and Kennedy pathway-related genes plays a vital role in lipid productivity in the oleaginous yeast L. starkeyi.

Fatty acid desaturases, especially Δ12 fatty acid desaturases, are key enzymes for the production of unsaturated fatty acids in oleaginous yeasts. In this study, we identified and characterized a gene encoding Δ12 fatty acid desaturase of Pseudozyma antarctica named PaFAD2. Almost all oleic acid (C18:1) was converted to linoleic acid by the heterologous expression of the PaFAD2 gene in Saccharomyces cerevisiae and Lipomyces starkeyi oleaginous yeast. Notably, PaFad2 converted not only oleic acid to linoleic acid, but also palmitoleic acid (C16:1) to 9,12-hexadecadienoic acid (C16:2). These results indicated that the PaFAD2 gene was very useful for the production of polyunsaturated fatty acids in yeast, including oleaginous yeast.

The oleaginous yeast Rhodosporodium toruloides is receiving widespread attention as an alternative energy source for biofuels due to its unicellular nature, high growth rate and because it can be fermented on a large-scale. In this study, R. toruloides was cultured under both light and dark conditions in order to understand the light response involved in lipid and carotenoid biosynthesis. Our results from phenotype and gene expression analysis showed that R. toruloides responded to light by producing darker pigmentation with an associated increase in carotenoid production. Whilst there was no observable difference in lipid production, slight changes in the fatty acid composition were recorded. Furthermore, a two-step response was found in three genes (GGPSI, CAR1, and CAR2) under light conditions and the expression of the gene encoding the photoreceptor CRY1 was similarly affected. Light effect on carotenoid production in Rhodosporidium toruloides

The oleaginous yeast Lipomyces starkeyi is a potential cost-effective source for the production of microbial lipids. Fatty acid elongases have vital roles in the syntheses of long-chain fatty acids. In this study, two genes encoding fatty acid elongases of L. starkeyi, LsELO1, and LsELO2 were identified and characterized. Heterologous expression of these genes in Saccharomyces cerevisiae revealed that LsElo1 is involved in the production of saturated long-chain fatty acids with 24 carbon atoms (C24:0) and that LsElo2 is involved in the conversion of C16 fatty acids to C18 fatty acids. In addition, both LsElo1 and LsElo2 were able to elongate polyunsaturated fatty acids. LsElo1 elongated linoleic acid (C18:2) to eicosadienoic acid (C20:2), and LsElo2 elongated α-linolenic acid (C18:3) to eicosatrienoic acid (C20:3). Overexpression of LsElo2 in L. starkeyi caused a reduction in C16 fatty acids, such as palmitic and palmitoleic acids, and an accumulation of C18 fatty acids such as oleic and linoleic acids. Our findings have the potential to contribute to the remodeling of fatty acid composition and the production of polyunsaturated long-chain fatty acids in oleaginous yeasts.

The plant defensin antifungal peptide 1 (AFP1) is composed of only 51 amino acids and has potent antifungal activity against the rice blast fungus Magnaporthe oryzae. To elucidate the antifungal mechanisms of AFP1, we assessed the suppression of appressorium formation and hyphal penetration by M. oryzae. AFP1 interfered with appressorium formation and host cell penetration following to invasion in a dose-dependent manner. This is the first report of the prevention of M. oryzae infection in rice by AFP1 and our observations could help to develop resistant cultivars and antifungal formulations for use in agriculture.

The oleaginous yeast Lipomyces starkeyi is an attractive organism for the industrial production of lipids; however, the amount of lipid produced by wild-type L. starkeyi is insufficient. The study aims to obtain L. starkeyi mutants that rapidly accumulate large amounts of triacylglycerol (TAG). Mutagenized yeast cells at the early stages of cultivation were subjected to Percoll density gradient centrifugation; cells with increased production of TAG were expected to be enriched in the resultant upper fraction because of their lower density. Among 120 candidates from the upper fractions, five mutants were isolated that accumulated higher amounts of TAG. Moreover, when omitting cells with mucoid colony morphology, 11 objective mutants from 11 candidates from the upper fraction were effectively (100%) isolated. Of total 16 mutants obtained, detailed characterization of five mutants was performed to reveal that five mutants achieved about 1.5-2.0 times TAG concentration (4.7-6.0 g/L) as compared with the wild-type strain (3.6 g/L) at day 5. Among these five mutants, strain E15 was the best for industrial use because only strain E15 showed significantly higher TAG concentration as well as significantly higher degree of lipid to glucose and biomass to glucose yields than the wild-type strain. Thus, Percoll density gradient centrifugation is an effective method to isolate mutant cells that rapidly accumulate large amounts of TAG. It is expected that by repeating this procedure as part of a yeast-breeding program, L. starkeyi mutants suitable for industrial lipid production can be easily and effectively obtained.

The oleaginous yeast Lipomyces starkeyi is an excellent producer of triacylglycerol (TAG) as a feedstock for biodiesel production. To understand the regulation of TAG synthesis, we attempted to isolate mutants with decreased lipid productivity and analyze the expression of TAG synthesis-related genes in this study. A mutant with greatly decreased lipid productivity, sr22, was obtained by an effective screening method using Percoll density gradient centrifugation. The expression of citrate-mediated acyl-CoA synthesis-related genes (ACL1, ACL2, ACC1, FAS1, and FAS2) was decreased in the sr22 mutant compared with that of the wild-type strain. Together with a notion that L. starkeyi mutants with increased lipid productivities had increased gene expression, there was a correlation between the expression of these genes and TAG synthesis. To clarify the importance of citrate-mediated acyl-CoA synthesis pathway on TAG synthesis, we also constructed a strain with no ATP-citrate lyase responsible for the first reaction of citrate-mediated acyl-CoA synthesis and investigated the importance of ATP-citrate lyase on TAG synthesis. The ATP-citrate lyase was required for the promotion of cell growth and TAG synthesis in a glucose medium. This study may provide opportunities for the development of an efficient TAG synthesis for biodiesel production.

Application of high hydrostatic pressure (HHP) treatment to food processing is expected as a non-thermal fermentation regulation technology that supresses over fermentation. However, the yeast Sacc...

The oleaginous yeast Lipomyces starkeyi is an excellent sustainable lipid producer, which can convert industrial wastes into lipids and accumulate triacylglycerols (TAG) by > 70% of its dry cell weight. Recent studies using omics technologies applied in L. starkeyi have aided in obtaining greater understanding of the important mechanisms of lipid metabolism in L. starkeyi. Therefore, the development of genetic engineering tools for L. starkeyi has led to accelerated efforts for a highly efficient production of lipids.This review focuses on the aspects of TAG and fatty acid synthesis pathways in L. starkeyi. We also present a quite effective strategy to obtain L. starkeyi mutants accumulating a larger amount of lipids and having a higher lipid production rate than the wild-type strain. The analysis of these mutants exhibiting high lipid production has led to the identification of important genes for achieving highly effective lipid production and thus advanced improvement in lipid production. Herein, our aim was to provide useful information to advance the development of L. starkeyi as a cost-effective TAG feedstock.Key Points•Oleaginous yeast Lipomyces starkeyi is an excellent sustainable lipid producer.•Efficient isolation of lipid-enriched L. starkeyi mutants depends on the low density of lipids.•Increased acyl-CoA synthesis pathway is important for improving lipid productivity.

The oleaginous yeast

Microbial lipids produced by oleaginous microorganisms as raw materials for the production of oleochemicals and biodiesel are sustainable while avoiding competition with food products. The oleaginous yeast Lipomyces starkeyi is an excellent lipid producer with a great industrial potential that is suitable as a valuable host to improve lipid production through genetic engineering modifications. However, genetic tools, including effective transformation methods, for L. starkeyi are insufficient for improvement of lipid production and analysis of lipid production mechanisms. We previously developed a polyethylene glycol (PEG)-mediated spheroplast transformation method that significantly improved the homologous recombination efficiency of L. starkeyi strain ∆lslig4. Although other transformation methods, including lithium acetate (LiAc)-mediated transformation and Agrobacterium tumefaciens-mediated transformation, have been reported, a more efficient and convenient transformation method for L. starkeyi is desired. In this study, we developed a novel electroporation transformation method that was first applied for integration of drug-resistance gene markers into the genome of L. starkeyi strain ∆lslig4 at the 18S ribosomal DNA locus of a multiple-copy gene, which yielded approximately 60 transformants/μg of DNA. Optimization of five parameters (i.e., cell growth phase, cell density, osmotic stabilizers, pretreatment agents, and electric conditions) enhanced the efficiency of transformation to approximately 1.5 × 104 transformants/μg of DNA. As compared with those of LiAc-mediated transformation and PEG-mediated spheroplast transformation, the efficiency of the proposed transformation method was increased by about 111- and 7-fold, respectively. Additionally, the transformation efficiency of our proposed electroporation method targeting a single-copy gene locus yielded 273 transformants/μg of DNA. To our knowledge, this is the first report of a successful electroporation method to accelerate analysis of lipid production by L. starkeyi.

Microbial lipids are sustainable feedstock for the production of oleochemicals and biodiesel. Oleaginous yeasts have recently been proposed as alternative lipid producers to plants and animals to promote sustainability in the chemical and fuel industries. The oleaginous yeast Lipomyces starkeyi has great industrial potential as an excellent lipid producer. However, improvement of its lipid productivity is essential for the cost-effective production of oleochemicals and fuels. Genetic and metabolic engineering of L. starkeyi via gene manipulation techniques may result in improvements in lipid production and our understanding of the mechanisms behind lipid biosynthesis pathways. We previously described an integrative transformation system using a drug-resistant marker for L. starkeyi. However, gene-targeting frequencies were very low because non-homologous recombination is probably predominant in L. starkeyi. Genetic engineering tools for L. starkeyi have not been sufficiently developed. In this study, we describe a new genetic tool and its application in L. starkeyi. To develop a highly efficient gene-targeting system for L. starkeyi, we constructed a series of mutants by disrupting genes for LsKu70p, LsKu80p, and/or LsLig4p, which share homology with other yeasts Ku70p, Ku80p, and Lig4p, respectively, being involved in non-homologous end-joining pathway. Deletion of the LsLIG4 gene dramatically improved the homologous recombination efficiency (80.0%) at the LsURA3 locus compared with that in the wild-type strain (1.4%), when 2000-bp homologous flanking regions were used. The homologous recombination efficiencies of the double mutant ∆l sku70∆lslig4 and the triple mutant ∆lsku70∆lsku80∆lslig4 were also markedly enhanced. Therefore, the L. starkeyi ∆lslig4 background strains have promise as efficient recipient strains for genetic and metabolic engineering approaches in this yeast.

Rice blast caused by Pyricularia oryzae is one of the most devastating diseases worldwide. This study aimed to investigate the antifungal activity of rice defensin OsAFP1 and its partial peptides against P. oryzae. The partial peptides near the N- and C-terminal regions of OsAFP1 exhibited approximately the same antifungal activity as the entire protein against P. oryzae. These partial peptides have the potential to be used as fungicides.

Rice blast caused by Pyricularia oryzae is one of the most devastating diseases worldwide. This study aimed to investigate the antifungal activity of rice defensin OsAFP1 and its partial peptides against P. oryzae. The partial peptides near the N- and C-terminal regions of OsAFP1 exhibited approximately the same antifungal activity as the entire protein against P. oryzae. These partial peptides have the potential to be used as fungicides.

2-Deoxy-scyllo-inosose (DOI) is the first intermediate in the 2-deoxystreptamine-containing aminoglycoside antibiotic biosynthesis pathway and has a six-membered carbocycle structure. DOI is a valuable material because it is easily converted to aromatic compounds and carbasugar derivatives. In this study, we isolated yeast strains capable of assimilating DOI as a carbon source. One of the strains, Cryptococcus podzolicus ND1, mainly converted DOI to scyllo-quercitol and (−)-vibo-quercitol, which is a valuable compound used as an antihypoglycemia agent and as a heat storage material. An NADH-dependent DOI reductase coding gene, DOIR, from C. podzolicus ND1 was cloned and successfully overexpressed in Escherichia coli. The purified protein catalyzed the irreversible reduction of DOI with NADH and converted DOI into (−)-vibo-quercitol. The enzyme had an optimal pH of 8.5 and optimal temperature of 35°C, respectively. The kcat of this enzyme was 9.98 s−1, and the Km values for DOI and NADH were 4.38 and 0.24 mM, respectively. The enzyme showed a strong preference for NADH and showed no activity with NADPH. Multiple-alignment analysis of DOI reductase revealed that it belongs to the GFO_IDH_MocA protein family and is an inositol dehydrogenase homolog in other fungi, such as Cryptococcus gattii, and bacteria, such as Bacillus subtilis. This is the first identification of a DOI-assimilating yeast and a gene involved in DOI metabolism in fungi.

Recently, we developed a new statistical method for revealing the regulatory systems in living cells. Our method is based on Structural Equation Modelling combined with factor analysis. In generally, Structural Equation Modelling is utilized to detect the model adaptability with the measured data, such as large-scale questionnaire data. In this study, we improved our developed iteration algorithm and gene selection procedure to infer the causalities between variables as a regulatory network from limited numerical data. Our improved gene selection method is based on cross correlation to summarize the time preceding information from gene expression profiles, which were systematically measured at 8 time points. Cross correlation is usually utilized as a measure of similarity between two waves by a time-lag application, and we defined the values of lags ranging from −2 to +4. By this improved method, we selected 14 genes as regulatory factors for the specific system in oleaginous yeast. In the inferred model, only 6 genes among the selected 14 genes were considered to affect the volume of oil accumulation in a closed and specific system. Our method will be useful to artificially control cell systems in the bioproduction and biotechnology fields.

An antifungal defensin, AFP1, of Brassica juncea inhibits the growth of various microorganisms. The molecular details of this inhibition remain largely unknown. Herein, we reveal that a specific structure of fungal sphingolipid glucosylceramide (GlcCer) is critical for the sensitivity of Candida albicans cells to AFP1. Our results revealed that AFP1 induces plasma membrane permeabilization and the production of reactive oxygen species (ROS) in wild-type C. albicans cells, but not in cells lacking the ninth methyl residue of the GlcCer sphingoid base moiety, which is a characteristic feature of fungi. AFP1-induced ROS production is responsible for its antifungal activity, with a consequent loss of yeast cell viability. These findings suggest that AFP1 specifically recognizes the structural difference of GlcCer for targeting of the fungal pathogens.

Some viruses exploit internal initiation for their propagation in the host cell. This type of initiation is facilitated by structured elements (internal ribosome entry site, IRES) upstream of the initiator AUG and requires only a reduced number of canonical initiation factors. An important example are IRES of the virus family Dicistroviridae that bind to the inter-subunit side of the small ribosomal 40S subunit and lead to the formation of elongation-competent 80S ribosomes without the help of any initiation factor. Here, we present a comprehensive functional and structural analysis of eukaryotic-specific ribosomal protein rpS25 in the context of this type of initiation and propose a structural model explaining the essential involvement of rpS25 for hijacking the ribosome.

A long form of tRNase Z (tRNase ZL) can cleave any target RNA at any desired site under the direction of artificial small guide RNA including approximately 25-nucleotide hook-shaped RNA. Here we show that human miR-103 can form a hook structure to guide target RNA cleavage by human cytosolic tRNase ZL in vitro. In vivo analyses using luciferase mRNAs modified to contain miR-103 target sequences in their 3' untranslated regions indicated that miR-103 downregulates gene expression through directing mRNA cleavage by tRNase ZL. The present data suggest the possibility that human cytosolic tRNase ZL modulates gene expression through a subset of microRNAs in the cells.

Pathogenic angiogenesis in various diseases including cancer, autoimmune diseases, and age-related macular degeneration is thought to be regressed with anti-angiogenic drugs. TRUE gene silencing is a new technology to eliminate a specific mRNA using synthetic sgRNA and cellular tRNase Z(L). To discover anti-angiogenic sgRNAs, we applied TRUE silencing to the VEGF gene. We examined eight sgRNAs for efficacy in targeting exogenous human VEGF mRNA. Many of them worked efficiently in 293 and HeLa cells. Two of them downregulated the endogenous VEGF gene expression in HeLa cells very efficiently, and the efficacy of these two sgRNAs surpassed that of siRNA extremely.

tRNase Z is an enzyme responsible for removing a 3' trailer from pre-tRNA. Although most tRNase Zs cleave pre-tRNAs immediately after the discriminator nucleotide with the exception of Thermotoga maritima tRNase Z, which cleaves after the (74)CCA(76) sequence, our knowledge was limited about how the cleavage site in pre-tRNA is selected. Bacterial tRNase Zs contain a unique domain termed flexible arm, which extends from the core domain. Using various tRNase Z variants, here we examined how the flexible arm affects the cleavage site selection. T. maritima tRNase Z variants with modified flexible arms shifted the cleavage site and a Bacillus subtilis tRNase Z variant with no flexible arm showed an anomalous cleavage activity. Some of the T. maritima/B. subtilis chimeric enzymes had both properties: they recognized (74)CCA(76)-containing pre-tRNA and cleaved it after the discriminator. Taken together, the present data indicate that the flexible arm is not essential for pre-tRNA binding but affects the cleavage site selection probably by pushing the distal region of the T arm in such a way that the discriminator nucleotide becomes closer to the catalytic site.

tRNA 3'-processing endoribonuclease (tRNase Z) is one of the enzymes involved in the 3'-end processing of precursor tRNAs and is a member of the metallo-beta-lactamase superfamily. tRNase Z crystal structures have revealed that the enzyme forms a dimer and has a characteristic domain, named a flexible arm or an exosite, which protrudes from the metallo-beta-lactamase core and is involved in tRNA binding. The refined structure of Thermotoga maritima tRNase Z has been determined at 1.97 A resolution, revealing the structure of the flexible arm and the zinc-bound active site. The structure of the flexible arm of T. maritima tRNase Z is distinct from those of the Bacillus subtilis and Escherichia coli tRNase Zs. A comparison of the three tRNase Z structures revealed differences in the dimer orientation, which may be related to the unique cleavage-site specificity of T. maritima tRNase Z.

2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.

Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature 3' termini. Its crystal structure has revealed a four-layer alphabeta/betaalpha sandwich fold that is typically found in the metallo-beta-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg2+ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn2+ ions restored the lost Mg2+-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.

The diversity and succession of microbial communities during the garbage composting with rice hull as an amendment were studied by denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal primers. Based on temperature changes, the composting process could be divided into thermophilic, cooling-down, and maturing stages. The DGGE profiles and clone library analysis revealed that the microbial community drastically changed during the composting process from the thermophilic to the maturing stages. The dominant bacterial group changed from the phylum Firmicutes in the thermophilic stage to the phylum Bacteroidetes in the maturing stage. This change in microbial communities may be significant for the composting process. The diversity of cultivated bacteria isolated from samples taken at various stages of the composting process was low. A total of 87 isolates were classified as belonging to only four different groups. These groups were also detected in the DGGE profiles and by the clone library analysis. Our study indicated that a combination of culture-dependent and -independent approaches could be very useful for monitoring both bacterial diversity and the succession of communities during the composting process. This study would be beneficial for assessing the ecological consequences of disposal of organic waste.

The tRNA 3′-processing endoribonuclease (tRNase Z or 3′ tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3′ trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.

tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from precursor tRNA (pre-tRNA). We purified approximately 85 kDa 3' tRNase from pig liver and determined its partial sequences. BLAST search of them suggested that the enzyme was the product of a candidate human prostate cancer susceptibility gene, ELAC2, the biological function of which was totally unknown. We cloned a human ELAC2 cDNA and expressed the ELAC2 protein in Escherichia coli. The recombinant ELAC2 was able to cleave human pre-tRNA(Arg) efficiently. The 3' tRNase activity of the yeast ortholog YKR079C was also observed. The C-terminal half of human ELAC2 was able to remove a 3' trailer from pre-tRNA(Arg), while the N-terminal half failed to do so. In the human genome exists a gene, ELAC1, which seems to correspond to the C-terminal half of 3' tRNase from ELAC2. We showed that human ELAC1 also has 3'-tRNase activity. Furthermore, we examined eight ELAC2 variants that seem to be associated with the occurrence of prostate cancer for 3'-tRNase activity. Seven ELAC2 variants which contain one to three amino acid substitutions showed efficient 3'-tRNase activities, while one truncated variant, which lacked a C-terminal half region, had no activity.

The oleaginous yeast, Lipomyces starkeyi, is an excellent lipid producer with great industrial potential. However, methods for molecular breeding have not been established for L. starkeyi. We describe the development of a system for targeted rDNA integration of multiple copies of a gene into L. starkeyi genome by spheroplast–polyethylene glycol transformation.

A long form (tRNase Z(L)) of tRNA 3' processing endoribonuclease (tRNase Z, or 3' tRNase) can cleave any target RNA at any desired site under the direction of artificial small guide RNA (sgRNA) that mimics a 5'-half portion of tRNA. Based on this enzymatic property, a gene silencing technology has been developed, in which a specific mRNA level can be downregulated by introducing into cells a synthetic 5'-half-tRNA that is designed to form a pre-tRNA-like complex with a part of the mRNA. Recently 5'-half-tRNA fragments have been reported to exist stably in various types of cells, although little is know about their physiological roles. We were curious to know if endogenous 5'-half-tRNA works as sgRNA for tRNase Z(L) in the cells. Here we show that human cytosolic tRNase Z(L) modulates gene expression through 5'-half-tRNA. We found that 5'-half-tRNA(Glu), which co-immunoprecipitates with tRNase Z(L), exists predominantly in the cytoplasm, functions as sgRNA in vitro, and downregulates the level of a luciferase mRNA containing its target sequence in human kidney 293 cells. We also demonstrated that the PPM1F mRNA is one of the genuine targets of tRNase Z(L) guided by 5'-half-tRNA(Glu). Furthermore, the DNA microarray data suggested that tRNase Z(L) is likely to be involved in the p53 signaling pathway and apoptosis.

In most organisms, tRNase Z is considered to be essential for 3' processing of tRNA molecules. The Escherichia coli tRNase Z gene, however, appears to be dispensable under normal growth conditions, and its existence remained an enigma. Here we intensively examined various (pre-)tRNAs for good substrates of E. coli tRNase Z in vitro, and found that the enzyme can remove the 3' terminal CCA residues from mature tRNAs regardless of their nucleotide modifications. Furthermore, we discovered that E. coli tRNase Z, when sufficiently expressed in the cell, can shut down growth probably by removing amino acids from aminoacyl-tRNAs. We confirmed in vitro that E. coli tRNase Z exceptionally possesses the activity that cleaves off the 3' terminal residues charging an amino acid from an aminoacyl-tRNA molecule. The current data suggest that tRNase Z might help modulate a cell growth rate by repressing translation under some stressful conditions.

We performed functional analyses for various single amino-acid substitution variants of Escherichia coli, Bacillus subtilis, and human tRNase Zs. The well-conserved six histidine, His(I)-His(VI), and two aspartate, Asp(I) and Asp(II), residues together with metal ions are thought to form the active site of tRNase Z. The Mn(2+)-rescue analysis for Thermotoga maritima tRNase Z(S) has suggested that Asp(I) and His(V) directly contribute the proton transfer for the catalysis, and a catalytic mechanism has been proposed. However, experimental evidence supporting the proposed mechanism was limited. Here we intensively examined E. coli and B. subtilis tRNase Z(S) variants and human tRNase Z(L) variants for cleavage activities on pre-tRNAs in the presence of Mg(2+) or Mn(2+) ions. We observed that the Mn(2+) ions cannot rescue the activities of Asp(I)Ala and His(V)Ala variants from each species, which are lost in the presence of Mg(2+). This observation may support the proposed catalytic mechanism.

A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.

We have been developing a unique system for the downregulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3'-processing endoribonuclease (tRNase Z(L)) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase Z(L) in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase Z(L) method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase Z(L) directed by sgRNA not owing to a simple antisense effect. We also show that tRNase Z(L) together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3beta by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.

tRNase Z, which exists in almost all cells, is believed to be working primarily for tRNA 3' maturation. In Escherichia coli, however, the tRNase Z gene appears to be dispensable under normal growth conditions, and its physiological role is not clear. Here, to investigate a possibility that E. coli tRNase Z cleaves RNAs other than pre-tRNAs, we tested several unstructured RNAs for cleavage. Surprisingly, all these substrates were cleaved very efficiently at multiple sites by a recombinant E. coli enzyme in vitro. tRNase Zs from Bacillus subtilis and Thermotoga maritima also cleaved various unstructured RNAs. The E. coli and B. subtilis enzymes seem to have a tendency to cleave after cytidine or before uridine, while cleavage by the T. maritima enzyme inevitably occurred after CCA in addition to the other cleavages. Assays to determine optimal conditions indicated that metal ion requirements differ between B. subtilis and T. maritima tRNase Zs. There was no significant difference in the observed rate constant between unstructured RNA and pre-tRNA substrates, while the K(d) value of a tRNase Z/unstructured RNA complex was much higher than that of an enzyme/pre-tRNA complex. Furthermore, eukaryotic tRNase Zs from yeast, pig, and human cleaved unstructured RNA at multiple sites, but an archaeal tRNase Z from Pyrobaculum aerophilum did not.

There exists a significant difference in pre-tRNA preference among prokaryotic tRNase Zs. This is an enigma, because pre-tRNAs should form the common L-shaped structure and tRNase Zs should form the common structure based on the αβ/βα-fold. To address this issue, we examined six different eubacterial and archaeal tRNase Zs including two newly isolated tRNase Zs for cleavage of 18 different pre-tRNA substrates. Two Thermotoga maritima , one Thermus thermophilus , one Bacillus subtilis , one Thermoplasma acidophilum , and one Pyrobaculum aerophilum enzymes were tested. To our surprise, the newly isolated proteins T. maritima TM0207 and T. thermophilus YP_145327 showed the weak tRNase Z activity, even though their primary amino acid sequences are, on the whole, quite different from those of the typical tRNase Zs. We confirmed that substrate recognition ability is quite different among those tRNase Zs. In addition, we found that the optimal conditions as a whole differ significantly among the enzymes. From these results, we provided several clues to solve the enigma by showing the potential importance of the 74th–76th nucleotide sequence of pre-tRNA, the flexible arm length of tRNase Z, the divalent metal ion species, and the histidine corresponding His222 in T. maritima tRNase Z.

tRNA 3'-processing endoribonucleases (tRNase Z, or 3'-tRNase; EC 3.1.26.11) are enzymes that remove 3'-trailers from pre-tRNAs. An about 12-base-pair stem, a T loop-like structure, and a 3'-trailer were considered to be the minimum requirements for recognition by the long form (tRNase ZL) of tRNase Z; tRNase ZL can recognize and cleave a micro-pre-tRNA or a hooker/target RNA complex that resembles a micro-pre-tRNA. We examined four hook RNAs containing systematically weakened T stems for directing target RNA cleavage by tRNase ZL. As expected, the cleavage efficiency decreased with the decrease in T stem stability, and to our surprise, even the hook RNA that forms no T stem-loop-directed slight cleavage of the target RNA, suggesting that the T stem-loop structure is important but dispensable for substrate recognition by tRNase ZL. To analyze the effect of the T loop on substrate recognition, we compared the cleavage reaction for a micro-pre-tRNA with that for a 12-base-pair double-stranded RNA, which is the same as the micro-pre-tRNA except for the lack of the T loop structure. The observed rate constant value for the double-stranded RNA was comparable with that for the micro-pre-tRNA, whereas the K(d) value for the complex with the double-stranded RNA was much higher than that for the complex with the micro-pre-tRNA. These results suggest that the T loop structure is not indispensable for the recognition, although the interaction between the T loop and the enzyme exists. Cleavage assays for such double-stranded RNA substrates of various lengths suggested that tRNase ZL can recognize and cleave double-stranded RNA substrates that are longer than 5 base pairs and shorter than 20 base pairs. We also showed that double-stranded RNA is not a substrate for the short form of tRNase Z.

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.

Transfer RNA (tRNA) 3' processing endoribonuclease (tRNase Z) is an enzyme responsible for the removal of a 3' trailer from pre-tRNA. There exists two types of tRNase Z: one is a short form (tRNase ZS) that consists of 300-400 amino acids, and the other is a long form (tRNase ZL) that contains 800-900 amino acids. Here we investigated whether the short and long forms have different preferences for various RNA substrates. We examined three recombinant tRNase ZSs from human, Escherichia coli and Thermotoga maritima, two recombinant tRNase ZLs from human and Saccharomyces cerevisiae, one tRNase ZL from pig liver, and the N- and C-terminal half regions of human tRNase ZL for cleavage of human micro-pre-tRNA(Arg) and the RNase 65 activity. All tRNase ZLs cleaved the micro-pre-tRNA and showed the RNase 65 activity, while all tRNase ZSs and both half regions of human tRNase ZL failed to do so with the exception of the C-terminal half, which barely cleaved the micro-pre-tRNA. We also show that only the long forms of tRNase Z can specifically cleave a target RNA under the direction of a new type of small guide RNA, hook RNA. These results indicate that indeed tRNase ZL and tRNase ZS have different substrate specificities and that the differences are attributed to the N-terminal half-domain of tRNase ZL. Furthermore, the optimal concentrations of NaCl, MgCl2 and MnCl2 differed between tRNase ZSs and tRNase ZLs, and the K(m) values implied that tRNase ZLs interact with pre-tRNA substrates more strongly than tRNase ZSs.

Cycloheximide (CYH) resistance in the yeast Candida maltosa is based on the inducible expression of genes encoding a variant of ribosomal protein L41-Q, with glutamine at position 56 instead of the proline found in normal L41. The promoter of L41-Q2a, one of the L41-Q gene alleles encoding L41-Q, has an element similar to the Gcn4p-responsive element of Saccharomyces cerevisiae. In a previous study, this element was shown to be essential for the induction of L41-Q by CYH. In the present study, a C. maltosa GCN4 homolog, C-GCN4, was cloned. It had a long 5′-leader region with three upstream open reading frames. Enhanced expression of the C-GCN4 reporter fusion gene upon the addition of 3-aminotriazole or by mutations in start codons of all three upstream open reading frames indicates that C-GCN4 expression is under translation repression as was seen with GCN4. The C-GCN4-depleted mutant was unable to grow in a nutrient medium containing CYH and did not express L41-Q genes. Recombinant C-Gcn4p bound to the consensus DNA element for Gcn4p, 5′-(G/A)TGACTCAT-3′, located upstream of L41-Q2a. Thus, C-Gcn4p, which likely functions in the general control of amino acid biosynthesis, is essential for the expression of L41-Q genes.

To clarify the mechanism of inducible resistance to cycloheximide (CYH) in Candida maltosa, where a transcriptional factor C-Gcn4p is expected to up-regulate the gene expression of the CYH-inducible resistant ribosomal protein L41.

The transcriptional activator protein Gcn4p increases the transcription of many genes that code for amino acid synthesis genes during amino acid starvation in Saccharomyces cerevisiae. Here we showed that after the disruption of C-GCN4, a homolog in Candida maltosa of GCN4 in S. cerevisiae, formed pseudohyphae in minimal medium. This is the first report that a GCN4 homolog is involved in the control of morphological transition.