Deletion of LsSNF1 enhances lipid accumulation in the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast, Lipomyces starkeyi can have diverse industrial applications due to its remarkable capacity to use various carbon sources for the biosynthesis intracellular triacylglycerides (TAGs). In L. starkeyi, TAG synthesis is enhanced through upregulation of genes involved in citrate-mediated acyl-CoA synthesis and Kennedy pathways through the transcriptional regulator LsSpt23p. High expression of LsSPT23 can considerably enhance TAG production. Altering the regulatory factors associated with lipid production can substantially augment lipid productivity. In this study, we identified and examined the L. starkeyi homolog sucrose nonfermenting 1 SNF1 (LsSNF1) of YlSNF1, which encodes a negative regulator of lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica. The deletion of LsSNF1 enhanced TAG productivity in L. starkeyi, suggesting that LsSnf1p is a negative regulator in TAG production. The enhancement of TAG production following deletion of LsSNF1 can primarily be attributed to the upregulation of genes in the citrate-mediated acyl-CoA synthesis and Kennedy pathways, pivotal routes in TAG biosynthesis. The overexpression of LsSPT23 enhanced lipid productivity; strain overexpressing LsSPT23 and without LsSNF1 exhibited increased TAG production capacity per cell. LsSnf1p also has a significant role in the utilization of carbon sources, including xylose or glycerol, in L. starkeyi. Our study results elucidated the role of LsSnf1p in the negative regulation of TAG synthesis in L. starkeyi, which has not previously been reported.

Optimization of the CRISPR/Cas9 system using adh1 promoter derivatives in fission yeast.

The CRSIPR/Cas9 system has been applied to fission yeast, but there remain some rooms for improvement. Here we report that the weaker versions of the adh1 + promoter, adh11 and adh41 promoters, for the potentially cytotoxic Cas9 achieved highly efficient mutagenesis and gene deletion at the ade6 + locus. Employing a drug-selectable marker instead of conventional auxotrophic markers, our new vector system is compatible with a variety of experimental settings including prototrophic/auxotrophic strains and complete/minimal media.

LsSpt23p is a regulator of triacylglycerol synthesis in the oleaginous yeast Lipomyces starkeyi.

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 POINTS: LsSpt23p was identified as a positive regulator of TAG biosynthesis LsSPT23 overexpression enhanced TAG biosynthesis gene expression and TAG production LsSPT23M1108T overexpression mutant showed fivefold higher TAG production than control.

Citrate-Mediated Acyl-CoA Synthesis Is Required for the Promotion of Growth and Triacylglycerol Production in Oleaginous Yeast Lipomyces starkeyi.

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.

Analysis of the light regulatory mechanism in carotenoid production in Rhodosporidium toruloides NBRC 10032.

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.

Isolation and characterization of Lipomyces starkeyi mutants with greatly increased lipid productivity following UV irradiation.

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.

Inference of Regulatory System for TAG Biosynthesis in Lipomyces starkeyi.

Improving the bioproduction ability of efficient host microorganisms is a central aim in bioengineering. To control biosynthesis in living cells, the regulatory system of the whole biosynthetic pathway should be clearly understood. In this study, we applied our network modeling method to infer the regulatory system for triacylglyceride (TAG) biosynthesis in Lipomyces starkeyi, using factor analyses and structural equation modeling to construct a regulatory network model. By factor analysis, we classified 89 TAG biosynthesis-related genes into nine groups, which were considered different regulatory sub-systems. We constructed two different types of regulatory models. One is the regulatory model for oil productivity, and the other is the whole regulatory model for TAG biosynthesis. From the inferred oil productivity regulatory model, the well characterized genes DGA1 and ACL1 were detected as regulatory factors. Furthermore, we also found unknown feedback controls in oil productivity regulation. These regulation models suggest that the regulatory factor induction targets should be selected carefully. Within the whole regulatory model of TAG biosynthesis, some genes were detected as not related to TAG biosynthesis regulation. Using network modeling, we reveal that the regulatory system is helpful for the new era of bioengineering.

Identification and characterization of Pseudozyma antarctica Δ12 fatty acid desaturase and its utilization for the production of polyunsaturated fatty acids.

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.

Lipid metabolism of the oleaginous yeast Lipomyces starkeyi.

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.

Effect of light on carotenoid and lipid production in the oleaginous yeast Rhodosporidium toruloides.

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.

Identification and characterization of two fatty acid elongases in Lipomyces starkeyi.

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.

A novel electroporation procedure for highly efficient transformation of Lipomyces starkeyi.

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.

Highly selective isolation and characterization of Lipomyces starkeyi mutants with increased production of triacylglycerol.

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.

Identification and characterization of Δ12 and Δ12/Δ15 bifunctional fatty acid desaturases in the oleaginous yeast Lipomyces starkeyi.

Fatty acid desaturases play vital roles in the synthesis of unsaturated fatty acids. In this study, Δ12 and Δ12/Δ15 fatty acid desaturases of the oleaginous yeast Lipomyces starkeyi, termed LsFad2 and LsFad3, respectively, were identified and characterized. Saccharomyces cerevisiae expressing LsFAD2 converted oleic acid (C18:1) to linoleic acid (C18:2), while a strain of LsFAD3-expressing S. cerevisiae converted oleic acid to linoleic acid, and linoleic acid to α-linolenic acid (C18:3), indicating that LsFad2 and LsFad3 were Δ12 and bifunctional Δ12/Δ15 fatty acid desaturases, respectively. The overexpression of LsFAD2 in L. starkeyi caused an accumulation of linoleic acid and a reduction in oleic acid levels. In contrast, overexpression of LsFAD3 induced the production of α-linolenic acid. Deletion of LsFAD2 and LsFAD3 induced the accumulation of oleic acid and linoleic acid, respectively. Our findings are significant for the commercial production of polyunsaturated fatty acids, such as ω-3 polyunsaturated fatty acids, in L. starkeyi.

Isolation of 2-deoxy-scyllo-inosose (DOI)-assimilating yeasts and cloning and characterization of the DOI reductase gene of Cryptococcus podzolicus ND1.

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.

Efficient gene targeting in non-homologous end-joining-deficient Lipomyces starkeyi strains.

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.

Partial peptides from rice defensin OsAFP1 exhibited antifungal activity against the rice blast pathogen Pyricularia oryzae.

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.

Multicopy integration and expression of heterologous genes in the oleaginous yeast, Lipomyces starkeyi.

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.

Antifungal activity of plant defensin AFP1 in Brassica juncea involves the recognition of the methyl residue in glucosylceramide of target pathogen Candida albicans.

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.

Structural basis for the binding of IRES RNAs to the head of the ribosomal 40S subunit.

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.