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Lipomyces starkeyi is an oleaginous yeast, and has been classified in four distinct groups, i.e., sensu stricto and custers α, β, and γ. Recently, L. starkeyi clusters α, β, and γ were recognized independent species, Lipomyces mesembrius, Lipomyces doorenjongii, and Lipomyces kockii, respectively. In this study, we investigated phylogenetic relationships within L. starkeyi, including 18 Japanese wild strains, and its related species, based on internal transcribed spacer sequences and evaluated biochemical characters which reflected the phylogenetic tree. Phylogenetic analysis showed that most of Japanese wild strains formed one clade and this clade is more closely related to L. starkeyi s.s. clade including one Japanese wild strain than other clades. Only three Japanese wild strains were genetically distinct from L. starkeyi. Lipomyces mesembrius and L. doorenjongii shared one clade, while L. kockii was genetically distinct from the other three species. Strains in L. starkeyi s.s. clade converted six sugars, D: -glucose, D: -xylose, L: -arabinose, D: -galactose, D: -mannose, and D: -cellobiose to produce high total lipid yields. The Japanese wild strains in subclades B, C, and D converted D: -glucose, D: -galactose, and D: -mannose to produce high total lipid yields. Lipomyces mesembrius was divided into two subclades. Lipomyces mesembrius CBS 7737 converted D: -xylose, L: -arabinose, D: -galactose, and D: -cellobiose, while the other L. mesembrius strains did not. Lipomyces doorenjongii converted all the sugars except D: -cellobiose. In comparison to L. starkeyi, L. mesembrius, and L. doorenjongii, L. kockii produced higher total lipid yields from D: -glucose, D: -galactose, and D: -mannose. The type of sugar converted depended on the subclade classification elucidated in this study.

The objective of this study was to disrupt the non-homologous end-joining (NHEJ) pathway gene (Ls ku70 Δ) and evaluate the effects of selected gene deletions related to glycogen synthesis (Ls GSY1 ) and lipid degradation (Ls MFE1 , Ls PEX10 , and Ls TGL4 ) on lipid production in the oleaginous yeast Lipomyces starkeyi . Disruption of the NHEJ pathway to reduce the rate of non-homologous recombination is a common approach used to overcome low-efficiency targeted deletion or insertion in various organisms. Here, the homologue of the Ls KU70 gene was identified and disrupted in L. starkeyi NRRL Y-11558. The Ls GSY1 , Ls MFE1 , Ls PEX10 , Ls TGL4 , and Ls URA3 genes were then replaced with a resistance marker in the Ls ku70 Δ strain and several site-specific insertions were assessed for targeted over-expression of selected genes. The targeted disruption efficiency of five selected genes (Ls GSY1 , Ls MFE1 , Ls PEX10 , Ls TGL4 , and Ls URA3 ) was increased from 0 to 10% in the parent to 50–100% of transformants screened in the Ls ku70 Δ strain with 0.8–1.4 kb homologous flanking sequences, while the efficiency of site-specific gene insertion with the β-glucuronidase reporter gene was 100% in the locus near the 3′-end coding (Ls KU70 ) and non-coding (Ls GSY1 , Ls MFE1 , and Ls PEX10 ) regions. Disruption of Ls KU70 in isolation and in conjunction with Ls GSY1 , Ls MFE1 , Ls PEX10 , or Ls TGL4 did not affect lipid production in L. starkeyi . Furthermore, β-glucuronidase reporter gene activity was similar in strains containing site-specific targeted insertions. Therefore, over-expression of genes related to lipid synthesis at targeted loci can be further examined for improvement of total lipid production in L. starkeyi .

Tall fescue (Schedonorus arundinaceus) is a widespread grass that can form a symbiotic relationship with a shoot-specific fungal endophyte (Epichloë coenophiala). While the effects of fungal endophyte infection on fescue physiology and ecology have been relatively well studied, less attention has been given to how this relationship may impact the soil microbial community. We used high-throughput DNA sequencing and phospholipid fatty acid analysis to determine the structure and biomass of microbial communities in both bulk and rhizosphere soils from tall fescue stands that were either uninfected with E. coenophiala or were infected with the common toxic strain or one of several novel strains of the endophyte. We found that rhizosphere and bulk soils harbored distinct microbial communities. Endophyte presence, regardless of strain, significantly influenced soil fungal communities, but endophyte effects were less pronounced in prokaryotic communities. E. coenophiala presence did not change total fungal biomass but caused a shift in soil and rhizosphere fungal community composition, increasing the relative abundance of taxa within the Glomeromycota phylum and decreasing the relative abundance of genera in the Ascomycota phylum, including Lecanicillium, Volutella, Lipomyces, Pochonia, and Rhizoctonia. Our data suggests that tripartite interactions exist between the shoot endophyte E. coenophiala, tall fescue, and soil fungi that may have important implications for the functioning of soils, such as carbon storage, in fescue-dominated grasslands.

A new mycovirus was found in the Fusarium culmorum strain A104-1 originally sampled on wheat in Belgium. This novel virus, for which the name Fusarium culmorum virus 1 (FcV1) is suggested, is phylogenetically related to members of the previously proposed family ‘’Unirnaviridae’’. FcV1 has a monopartite dsRNA genome of 2898 bp that harbors two large non-overlapping ORFs. A typical -1 slippery motif is found at the end of ORF1, advocating that ORF2 is translated by programmed ribosomal frameshifting. While ORF2 exhibits a conserved replicase domain, ORF1 encodes for an undetermined protein. Interestingly, a hypothetically transcribed gene similar to unirnaviruses ORF1 was found in the genome of Lipomyces starkeyi, presumably resulting from a viral endogenization in this yeast. Conidial isolation and chemical treatment were unsuccessful to obtain a virus-free isogenic line of the fungal host, highlighting a high retention rate for FcV1 but hindering its biological characterization. In parallel, attempt to horizontally transfer FcV1 to another strain of F. culmorum by dual culture failed. Eventually, a screening of other strains of the same fungal species suggests the presence of FcV1 in two other strains from Europe.

The oleaginous yeast Lipomyces starkeyi has considerable potential in industrial application, since it can accumulate a large amount of triacylglycerol (TAG), which is produced from sugars under nitrogen limitation condition. However, the regulation of lipogenesis in L. starkeyi has not been investigated in depth. In this study, we compared the genome sequences of wild-type and mutants with increased TAG productivity, and identified a regulatory protein, LsSpt23p, which contributes to the regulation of TAG synthesis in L. starkeyi. L. starkeyi mutants overexpressing LsSPT23 had increased TAG productivity compared with the wild-type strain. Quantitative real-time PCR analysis showed that LsSpt23p upregulated the expression of GPD1, which encodes glycerol 3-phosphate dehydrogenase; the Kennedy pathway genes SCT1, SLC1, PAH1, DGA1, and DGA2; the citrate-mediated acyl-CoA synthesis pathway-related genes ACL1, ACL2, ACC1, FAS1, and FAS2; and OLE1, which encodes ∆9 fatty acid desaturase. Chromatin immunoprecipitation-quantitative PCR assays indicated that LsSpt23p acts as a direct regulator of SLC1 and PAH1, all the citrate-mediated acyl-CoA synthesis pathway–related genes, and OLE1. These results indicate that LsSpt23p regulates TAG synthesis. Phosphatidic acid is a common substrate of phosphatidic acid phosphohydrolase, which is used for TAG synthesis, and phosphatidate cytidylyltransferase 1 for phospholipid synthesis in the Kennedy pathway. LsSpt23p directly regulated PAH1 but did not affect the expression of CDS1, suggesting that the preferred route of carbon is the Pah1p-mediated TAG synthesis pathway under nitrogen limitation condition. The present study contributes to understanding the regulation of TAG synthesis, and will be valuable in future improvement of TAG productivity in oleaginous yeasts.Key pointsLsSpt23p was identified as a positive regulator of TAG biosynthesisLsSPT23 overexpression enhanced TAG biosynthesis gene expression and TAG productionLsSPT23M1108Toverexpression mutant showed fivefold higher TAG production than control

Background Lipomyces starkeyi has been widely regarded as a promising oleaginous yeast with broad industrial application prospects because of its wide substrate spectrum, good adaption to fermentation inhibitors, excellent fatty acid composition for high-quality biodiesel, and negligible lipid remobilization. However, the currently low experimental lipid yield of L. starkeyi prohibits its commercial success. Metabolic model is extremely valuable to comprehend the complex biochemical processes and provide great guidance for strain modification to facilitate the lipid biosynthesis. Results A small-scale metabolic model of L. starkeyi NRRL Y-11557 was constructed based on the genome annotation information. The theoretical lipid yields of glucose, cellobiose, xylose, glycerol, and acetic acid were calculated according to the flux balance analysis (FBA). The optimal flux distribution of the lipid synthesis showed that pentose phosphate pathway (PPP) independently met the necessity of NADPH for lipid synthesis, resulting in the relatively low lipid yields. Several targets (NADP-dependent oxidoreductases) beneficial for oleaginicity of L. starkeyi with significantly higher theoretical lipid yields were compared and elucidated. The combined utilization of acetic acid and other carbon sources and a hypothetical reverse β-oxidation (RBO) pathway showed outstanding potential for improving the theoretical lipid yield. Conclusions The lipid biosynthesis potential of L. starkeyi can be significantly improved through appropriate modification of metabolic network, as well as combined utilization of carbon sources according to the metabolic model. The prediction and analysis provide valuable guidance to improve lipid production from various low-cost substrates.

Abstract Phylogenetic relationships among species assigned to genera of the family Lipomycetaceae were determined from analysis of the nearly entire large, subunit rRNA gene, the small subunit rRNA gene, mitochondrial small subunit rRNA gene and the translation elongation factor-1α gene. Monophyly of the Lipomycetaceae was strongly supported, and currently described species appear genetically unique. The multigene analysis provided no support for maintaining the genera Kawasakia, Smithiozyma, Waltomyces or Zygozyma, and it is proposed that species in these genera be assigned to the genus Lipomyces. The monotypic genus Babjevia is a member of the Dipodascopsis clade and it is proposed to reassign Babjevia anomala to Dipodascopsis. The proposed changes will result in the Lipomycetaceae having two ascosporic genera, Lipomyces and Dipodascopsis, and the anamorphic genus Myxozyma.

Lipomyces starkeyi is known to be associated with three strains-clusters showing high mutual nDNA reassociation within each cluster, but which reassociate ambiguously with the type of L. starkeyi. Representative strains of L. starkeyi and Cluster alpha were examined for possible genetic exchange by the prototrophic selection technique. Since no genetic recombination was detected, the strains are presumed to be genetically isolated. Cluster alpha is consequently assigned to the rank of species as Lipomyces mesembrius. A description of the new species is given. Lipomyces kononenkoae ssp. spencermartinsiae has been raised to the rank of species as L. spencermartinsiae.

Phylogenetic relationships of the yeast genus Lipomyces were studied using sequences from fragments of 5.8S rRNA gene and from internal transcribed spacer region ITS2 of 13 strains (7 type strains included) representing five species and subtaxa, and originating from different geographical locations (Japan, Trinidad, Nigeria, North America, Western Europe, Russia, South Africa, Mauritius). Parsimony and distance analyses were performed. Tree topology from the parsimony and distance analyses of the sequence confirmed the results of nDNA reassociation. Results segregate the 13 isolates of Lipomyces into five major clades.