Structure of the SigF1-dependent pilA1 gene promoter and characterization of the light-activated response in the cyanobacterium Synechococcus elongatus PCC 7942.

In cyanobacteria that perform oxygenic photosynthesis, alternative sigma factors can play critical roles in environmental acclimation at the transcriptional initiation step. Here, we found in Synechococcus elongatus PCC 7942 that transcription of the pilA1 gene, encoding the type IV pilin, is dependent on one of the group 3 sigma factors, SigF1. We analyzed the promoter sequence determinants and proposed herein that the -10 and -35 boxes upstream of the transcriptional start site are critical for transcription. Interestingly, while the pilA1 promoter is activated by illumination, RNA polymerase containing SigF1 is already located on the promoter region under dark conditions, prior to illumination. This strongly suggests that promoter activation by light follows the recruitment of RNA polymerase during transcriptional initiation.

Expression of bacterial phosphite dehydrogenase confers phosphite availability in a unicellular red alga Cyanidioschyzon merolae.

　Microalgae are promising cell factories for producing value-added products. Large-scale microalgal cultivation suffers from invasion by contaminating microorganisms. Since most contaminating organisms cannot utilize phosphite as a unique phosphorus source, phosphite-utilizing ability may provide a growth advantage against contaminating organisms and solve this problem. Studies showed that microorganisms, typically unable to metabolize phosphite, can utilize phosphite by expressing exogenous phosphite dehydrogenase. Here, we constructed Cyanidioschyzon merolae strains introduced with the phosphite dehydrogenase gene, ptxD, from Ralstonia sp. 4506. The ptxD-introduced strains grew in a phosphite-dependent manner, with the phosphite-related growth rate almost matching that with phosphate as sole phosphorus source.

DnaK2 Mediates a Negative Feedback Regulation of the Heat Shock Responsive Hik2-Rre1 Two-Component System in the Cyanobacterium Synechococcus Elongatus PCC 7942.

The two-component system (TCS) is a conserved signal transduction module in bacteria. The Hik2-Rre1 system is responsible for transcriptional activation upon high-temperature shift as well as plastoquinone-related redox stress in the cyanobacterium Synechococcus elongatus PCC 7942. As heat-induced de novo protein synthesis was previously shown to be required to quench the heat-activated response, we investigated the underlying mechanism in this study. We found that the heat-inducible transcription activation was alleviated by the overexpression of dnaK2, which is an essential homolog of the highly conserved HSP70 chaperone and whose expression is induced under the control of the Hik2-Rre1 TCS. Phosphorylation of Rre1 correlated with transcription of the regulatory target hspA. The redox stress response was found to be similarly repressed by dnaK2 overexpression. Considered together with the previous information, we propose a negative feedback mechanism of the Hik2-Rre1-dependent stress response that maintains the cellular homeostasis mediated by DnaK2.

ppGpp accumulation reduces the expression of the global nitrogen homeostasis-modulating NtcA regulon by affecting 2-oxoglutarate levels.

The cyanobacterium Synechococcus elongatus PCC 7942 accumulates alarmone guanosine tetraphosphate (ppGpp) under stress conditions, such as darkness. A previous study observed that artificial ppGpp accumulation under photosynthetic conditions led to the downregulation of genes involved in the nitrogen assimilation system, which is activated by the global nitrogen regulator NtcA, suggesting that ppGpp regulates NtcA activity. However, the details of this mechanism have not been elucidated. Here, we investigate the metabolic responses associated with ppGpp accumulation by heterologous expression of the ppGpp synthetase RelQ. The pool size of 2-oxoglutarate (2-OG), which activates NtcA, is significantly decreased upon ppGpp accumulation. De novo 13C-labeled CO2 assimilation into the Calvin-Benson-Bassham cycle and glycolytic intermediates continues irrespective of ppGpp accumulation, whereas the labeling of 2-OG is significantly decreased under ppGpp accumulation. The low 2-OG levels in the RelQ overexpression cells could be because of the inhibition of metabolic enzymes, including aconitase, which are responsible for 2-OG biosynthesis. We propose a metabolic rearrangement by ppGpp accumulation, which negatively regulates 2-OG levels to maintain carbon and nitrogen balance.

Label-free visualization of photosynthetic microbial biofilms using mid-infrared photothermal and autofluorescence imaging.

The formation of photosynthetic microbial biofilms comprising multispecies biomolecules, such as extracellular polymeric substances (EPSs), and microbial cells play pivotal roles in maintaining or stimulating their biological functions. Although there are numerous studies on photosynthetic microbial biofilms, the spatial distribution of EPS components that are vital for microbial biofilm formation, such as exopolysaccharides and proteins, is not well understood. Visualization of photosynthetic microbial biofilms requires label-free methods, because labelling EPSs results in structural changes or aggregation. Raman spectroscopy is useful for label-free visualization of biofilm constituents based on chemical contrast. However, interference resulting from the bright autofluorescence of photosynthetic molecules and the low detection efficiency of Raman scattering make visualization a challenge. Herein, we visualized photosynthetic microbial biofilms in a label-free manner using a super-resolution optical infrared absorption imaging technique, called mid-infrared photothermal (MIP) microscopy. By leveraging the advantages of MIP microscopy, such as its sub-micrometer spatial resolution, autofluorescence-free features, and high detection sensitivity, the distribution of cyanobacteria and their extracellular polysaccharides in the biofilm matrix were successfully visualized. This showed that cyanobacterial cells were aligned along acidic/sulfated polysaccharides in the extracellular environment. Furthermore, spectroscopic analyses elucidated that during formation of biofilms, sulfated polysaccharides initially form linear structures followed by entrapment of cyanobacterial cells. The present study provides the foundation for further studies on the formation, structure, and biological functions of microbial biofilms.

Molecular Bulkiness of a Single Amino Acid in the F1 α-Subunit Determines the Robustness of Cyanobacterial ATP Synthase.

Cyanobacteria are promising photosynthetic organisms owing to their ease of genetic manipulation. Among them, Synechococcus elongatus UTEX 2973 exhibits faster growth, higher biomass production efficiency and more robust stress tolerance compared with S. elongatus PCC 7942. This is due to specific genetic differences, including four single-nucleotide polymorphisms (SNPs) in three genes. One of these SNPs alters an amino acid at position 252 of the FoF1 ATP synthase α-subunit from Tyr to Cys (αY252C) in S. elongatus 7942. This change has been shown to significantly affect growth rate and stress tolerance, specifically in S. elongatus. Furthermore, experimental substitutions with several other amino acids have been shown to alter the ATP synthesis rate in the cell. In the present study, we introduced identical amino acid substitutions into Synechocystis sp. PCC 6803 at position 252 to elucidate the amino acid's significance and generality across cyanobacteria. We investigated the resulting impact on growth, intracellular enzyme complex levels, intracellular ATP levels and enzyme activity. The results showed that the αY252C substitution decreased growth rate and high-light tolerance. This indicates that a specific bulkiness of this amino acid's side chain is important for maintaining cell growth. Additionally, a remarkable decrease in the membrane-bound enzyme complex level was observed. However, the αY252C substitution did not affect enzyme activity or intracellular ATP levels. Although the mechanism of growth suppression remains unknown, the amino acid at position 252 is expected to play an important role in enzyme complex formation.

Coordination of apicoplast transcription in a malaria parasite by internal and host cues.

The malaria parasite Plasmodium falciparum has a nonphotosynthetic plastid called the apicoplast, which contains its own genome. Regulatory mechanisms for apicoplast gene expression remain poorly understood, despite this organelle being crucial for the parasite life cycle. Here, we identify a nuclear-encoded apicoplast RNA polymerase σ subunit (sigma factor) which, along with the α subunit, appears to mediate apicoplast transcript accumulation. This has a periodicity reminiscent of parasite circadian or developmental control. Expression of the apicoplast subunit gene, apSig, together with apicoplast transcripts, increased in the presence of the blood circadian signaling hormone melatonin. Our data suggest that the host circadian rhythm is integrated with intrinsic parasite cues to coordinate apicoplast genome transcription. This evolutionarily conserved regulatory system might be a future target for malaria treatment.

Low-temperature and circadian signals are integrated by the sigma factor SIG5.

Chloroplasts are a common feature of plant cells and aspects of their metabolism, including photosynthesis, are influenced by low-temperature conditions. Chloroplasts contain a small circular genome that encodes essential components of the photosynthetic apparatus and chloroplast transcription/translation machinery. Here, we show that in Arabidopsis, a nuclear-encoded sigma factor that controls chloroplast transcription (SIGMA FACTOR5) contributes to adaptation to low-temperature conditions. This process involves the regulation of SIGMA FACTOR5 expression in response to cold by the bZIP transcription factors ELONGATED HYPOCOTYL5 and ELONGATED HYPOCOTYL5 HOMOLOG. The response of this pathway to cold is gated by the circadian clock, and it enhances photosynthetic efficiency during long-term cold and freezing exposure. We identify a process that integrates low-temperature and circadian signals, and modulates the response of chloroplasts to low-temperature conditions.

A high-temperature sensitivity of Synechococcus elongatus PCC 7942 due to a tRNA-Leu mutation.

Certain mutations of the model cyanobacterium Synechococcus elongatus PCC 7942 during laboratory storage have resulted in some divergent phenotypes. One laboratory-stored strain (H1) shows a temperature-sensitive (ts) growth phenotype at 40 °C. Here, we investigated the reason for this temperature sensitivity. Whole genome sequencing of H1 identified a single nucleotide mutation in synpcc7942_R0040 encoding tRNA-Leu(CAA). The mutation decreases the length of the tRNA-Leu t-arm from 5 to 4 base pairs, and this explains the ts phenotype. Secondary mutations suppressing the ts phenotype were identified in synpcc7942_1640, which putatively encodes a NYN domain-containing protein (nynA). The NYN domain is thought to be involved in tRNA/rRNA degradation. Thus, the structural stability of tRNA-Leu is critical for growth at 40 °C in Synechococcus elongatus PCC 7942.

Pathogenesis-related protein 1 suppresses oomycete pathogen by targeting against AMPK kinase complex.

INTRODUCTION: During the arms race between plants and pathogens, pathogenesis-related proteins (PR) in host plants play a crucial role in disease resistance, especially PR1. PR1 constitute a secretory peptide family, and their role in plant defense has been widely demonstrated in both hosts and in vitro. However, the mechanisms by which they control host-pathogen interactions and the nature of their targets within the pathogen remain poorly understood. OBJECTIVES: The present study was aimed to investigate the anti-oomycete activity of secretory PR1 proteins and elaborate their underlying mechanisms. METHODS: This study was conducted in the potato-Phytophthora infestans pathosystem. After being induced by the pathogen infection, the cross-kingdom translocation of secretory PR1 was demonstrated by histochemical assays and western blot, and their targets in P. infestans were identified by yeast-two-hybrid assays, bimolecular fluorescence complementation assays, and co-immunoprecipitation assay. RESULTS: The results showed that the expression of secretory PR1-encoding genes was induced during pathogen infection, and the host could deliver PR1 into P. infestans to inhibit its vegetative growth and pathogenicity. The translocated secretory PR1 targeted the subunits of the AMPK kinase complex in P. infestans, thus affecting the AMPK-driven phosphorylation of downstream target proteins, preventing ROS homeostasis, and down-regulating the expression of RxLR effectors. CONCLUSION: The results provide novel insights into the molecular function of PR1 in protecting plants against pathogen infection, and uncover a potential target for preventing pre- and post-harvest late blight.

The Sulfide-Responsive SqrR/BigR Homologous Regulator YgaV of Escherichia coli Controls Expression of Anaerobic Respiratory Genes and Antibiotic Tolerance.

Compositions and activities of bacterial flora in the gastrointestinal tract significantly influence the metabolism, health, and disease of host humans and animals. These enteric bacteria can switch between aerobic and anaerobic growth if oxygen tension becomes limited. Interestingly, the switching mechanism is important for preventing reactive oxygen species (ROS) production and antibiotic tolerance. Studies have also shown that intracellular and extracellular sulfide molecules are involved in this switching control, although the mechanism is not fully clarified. Here, we found that YgaV, a sulfide-responsive transcription factor SqrR/BigR homolog, responded to sulfide compounds in vivo and in vitro to control anaerobic respiratory gene expression. YgaV also responded to H2O2 scavenging in the enteric bacterium Escherichia coli. Although the wild-type (WT) showed increased antibiotic tolerance under H2S-atmospheric conditions, the ygaV mutant did not show such a phenotype. Additionally, antibiotic sensitivity was higher in the mutant than in the WT of both types in the presence and absence of exogenous H2S. These results, therefore, indicated that YgaV-dependent transcriptional regulation was responsible for maintaining redox homeostasis, ROS scavenging, and antibiotic tolerance.

Constitutive glucose dehydrogenase elevates intracellular NADPH levels and luciferase luminescence in Bacillus subtilis.

BACKGROUND: Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer's disease. This conversion comprises two reactions catalyzed by two distinct inositol dehydrogenases, IolG and IolW. The IolW-mediated reaction requires the intracellular regeneration of NADPH, and there appears to be a limit to the endogenous supply of NADPH, which may be one of the rate-determining factors for the conversion of inositol. The primary mechanism of NADPH regeneration in this bacterium remains unclear. RESULTS: The gdh gene of B. subtilis encodes a sporulation-specific glucose dehydrogenase that can use NADP+ as a cofactor. When gdh was modified to be constitutively expressed, the intracellular NADPH level was elevated, increasing the conversion of inositol. In addition, the bacterial luciferase derived from Photorhabdus luminescens became more luminescent in cells in liquid culture and colonies on culture plates. CONCLUSION: The results indicated that the luminescence of luciferase was representative of intracellular NADPH levels. Luciferase can therefore be employed to screen for mutations in genes involved in NADPH regeneration in B. subtilis, and artificial manipulation to enhance NADPH regeneration can promote the production of substances such as scyllo-inositol.

The ferredoxin/thioredoxin pathway constitutes an indispensable redox-signaling cascade for light-dependent reduction of chloroplast stromal proteins.

To ensure efficient photosynthesis, chloroplast proteins need to be flexibly regulated under fluctuating light conditions. Thiol-based redox regulation plays a key role in reductively activating several chloroplast proteins in a light-dependent manner. The ferredoxin (Fd)/thioredoxin (Trx) pathway has long been recognized as the machinery that transfers reducing power generated by photosynthetic electron transport reactions to redox-sensitive target proteins; however, its biological importance remains unclear, because the complete disruption of the Fd/Trx pathway in plants has been unsuccessful to date. Especially, recent identifications of multiple redox-related factors in chloroplasts, as represented by the NADPH-Trx reductase C, have raised a controversial proposal that other redox pathways work redundantly with the Fd/Trx pathway. To address these issues directly, we used CRISPR/Cas9 gene editing to create Arabidopsis mutant plants in which the activity of the Fd/Trx pathway was completely defective. The mutants generated showed severe growth inhibition. Importantly, these mutants almost entirely lost the ability to reduce several redox-sensitive proteins in chloroplast stroma, including four Calvin-Benson cycle enzymes, NADP-malate dehydrogenase, and Rubisco activase, under light conditions. These striking phenotypes were further accompanied by abnormally developed chloroplasts and a drastic decline in photosynthetic efficiency. These results indicate that the Fd/Trx pathway is indispensable for the light-responsive activation of diverse stromal proteins and photoautotrophic growth of plants. Our data also suggest that the ATP synthase is exceptionally reduced by other pathways in a redundant manner. This study provides an important insight into how the chloroplast redox-regulatory system operates in vivo.

Glutamine synthetase mRNA releases sRNA from its 3'UTR to regulate carbon/nitrogen metabolic balance in Enterobacteriaceae.

Glutamine synthetase (GS) is the key enzyme of nitrogen assimilation induced under nitrogen limiting conditions. The carbon skeleton of glutamate and glutamine, 2-oxoglutarate, is supplied from the TCA cycle, but how this metabolic flow is controlled in response to nitrogen availability remains unknown. We show that the expression of the E1o component of 2-oxoglutarate dehydrogenase, SucA, is repressed under nitrogen limitation in Salmonella enterica and Escherichia coli. The repression is exerted at the post-transcriptional level by an Hfq-dependent sRNA GlnZ generated from the 3'UTR of the GS-encoding glnA mRNA. Enterobacterial GlnZ variants contain a conserved seed sequence and primarily regulate sucA through base-pairing far upstream of the translation initiation region. During growth on glutamine as the nitrogen source, the glnA 3'UTR deletion mutants expressed SucA at higher levels than the S. enterica and E. coli wild-type strains, respectively. In E. coli, the transcriptional regulator Nac also participates in the repression of sucA. Lastly, this study clarifies that the release of GlnZ from the glnA mRNA by RNase E is essential for the post-transcriptional regulation of sucA. Thus, the mRNA coordinates the two independent functions to balance the supply and demand of the fundamental metabolites.

A distinct class of GTP-binding proteins mediates chloroplast protein import in Rhodophyta.

Chloroplast protein import is mediated by translocons named TOC and TIC on the outer and inner envelope membranes, respectively. Translocon constituents are conserved among green lineages, including plants and green algae. However, it remains unclear whether Rhodophyta (red algae) share common chloroplast protein import mechanisms with the green lineages. We show that in the rhodophyte Cyanidioschyzon merolae, plastome-encoded Tic20pt localized to the chloroplast envelope and was transiently associated with preproteins during import, suggesting its conserved function as a TIC constituent. Besides plastome-encoded FtsHpt and several chaperones, a class of GTP (guanosine 5'-triphosphate)-binding proteins distinct from the Toc34/159 GTPase family associated transiently with preproteins. This class of proteins resides mainly in the cytosol and shows sequence similarities with Sey1/RHD3, required for endoplasmic reticulum membrane fusion, and with the periplastid-localized import factor PPP1, previously identified in the Apicomplexa and diatoms. These GTP-binding proteins, named plastid targeting factor for protein import 1 (PTF1) to PTF3, may act as plastid targeting factors in Rhodophyta.

CmNDB1 and a Specific Domain of CmMYB1 Negatively Regulate CmMYB1-Dependent Transcription of Nitrate Assimilation Genes Under Nitrogen-Repleted Condition in a Unicellular Red Alga.

Nitrogen assimilation is an essential process that controls plant growth and development. Plant cells adjust the transcription of nitrogen assimilation genes through transcription factors (TFs) to acclimatize to changing nitrogen levels in nature. However, the regulatory mechanisms of these TFs under nitrogen-repleted (+N) conditions in plant lineages remain largely unknown. Here, we identified a negative domain (ND) of CmMYB1, the nitrogen-depleted (-N)-activated TF, in a unicellular red alga Cyanidioschyzon merolae. The ND deletion changed the localization of CmMYB1 from the cytoplasm to the nucleus, enhanced the binding efficiency of CmMYB1 to promoters of nitrate assimilation genes, and increased the transcripts of nitrate assimilation genes under +N condition. A pull-down assay using an ND-overexpressing strain combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis helped us to screen and identify an unknown-function protein, the CmNDB1. Yeast two-hybrid analysis demonstrated that CmNDB1 interacts with ND. Similar to ND deletion, CmNDB1 deletion also led to the nucleus localization of CmMYB1, enhanced the promoter-binding ratio of CmMYB1 to the promoter regions of nitrate assimilation genes, and increased transcript levels of nitrate assimilation genes under +N condition. Thus, these presented results indicated that ND and CmNDB1 negatively regulate CmMYB1 functions under the +N condition in C. merolae.

Conserved Two-component Hik2-Rre1 Signaling Is Activated Under Temperature Upshift and Plastoquinone-reducing Conditions in the Cyanobacterium Synechococcus elongatus PCC 7942.

The highly conserved Hik2-Rre1 two-component system is a multi-stress responsive signal-transducing module that controls the expression of hsp and other genes in cyanobacteria. Previously, we found in Synechococcus elongatus PCC 7942 that the heat-inducible phosphorylation of Rre1 was alleviated in a hik34 mutant, suggesting that Hik34 positively regulates signaling. In this study, we examined the growth of the hik34 deletion mutant in detail, and newly identified suppressor mutations located in rre1 or sasA gene negating the phenotype. Subsequent analyses indicated that heat-inducible Rre1 phosphorylation is dependent on Hik2 and that Hik34 modulates this Hik2-dependent response. In the following part of this study, we focused on the mechanism to control the Hik2 activity. Other recent studies reported that Hik2 activity is regulated by the redox status of plastoquinone (PQ) through the 3Fe-4S cluster attached to the cyclic GMP, adenylyl cyclase, FhlA (GAF) domain. Consistent with this, Rre1 phosphorylation occurred after the addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone but not after the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea to the culture medium, which corresponded to PQ-reducing or -oxidizing conditions, respectively, suggesting that the Hik2-to-Rre1 phosphotransfer was activated under PQ-reducing conditions. However, there was no correlation between the measured PQ redox status and Rre1 phosphorylation during the temperature upshift. Therefore, changes in the PQ redox status are not the direct reason for the heat-inducible Rre1 phosphorylation, while some redox regulation is likely involved as oxidation events dependent on 2,6-dichloro-1,4-benzoquinone prevented heat-inducible Rre1 phosphorylation. On the basis of these results, we propose a model for the control of Hik2-dependent Rre1 phosphorylation.

Autofluorescence-based high-throughput isolation of nonbleaching Cyanidioschyzon merolae strains under nitrogen-depletion.

Photosynthetic organisms maintain optimum levels of photosynthetic pigments in response to environmental changes to adapt to the conditions. The identification of cyanobacteria strains that alleviate bleaching has revealed genes that regulate levels of phycobilisome, the main light-harvesting complex. In contrast, the mechanisms of pigment degradation in algae remain unclear, as no nonbleaching strains have previously been isolated. To address this issue, this study attempted to isolate nonbleaching strains of the unicellular red alga Cyanidioschyzon merolae after exposure to nitrogen (N)-depletion based on autofluorescence information. After four weeks under N-depletion, 13 cells from 500,000 cells with almost identical pre- and post-depletion chlorophyll a (Chl a) and/or phycocyanin autofluorescence intensities were identified. These nonbleaching candidate strains were sorted via a cell sorter, isolated on solid medium, and their post-N-depletion Chl a and phycocyanin levels were analyzed. Chl a levels of these nonbleaching candidate strains were lower at 1-4 weeks of N-depletion similar to the control strains, however, their phycocyanin levels were unchanged. Thus, we successfully isolated nonbleaching C. merolae strains in which phycocyanin was not degraded under N-depletion, via autofluorescence spectroscopy and cell sorting. This versatile method will help to elucidate the mechanisms regulating pigments in microalgae.

The Unicellular Red Alga Cyanidioschyzon merolae, an Excellent Model Organism for Elucidating Fundamental Molecular Mechanisms and Their Applications in Biofuel Production.

Microalgae are considered one of the best resources for the production of biofuels and industrially important compounds. Various models have been developed to understand the fundamental mechanism underlying the accumulation of triacylglycerols (TAGs)/starch and to enhance its content in cells. Among various algae, the red alga Cyanidioschyzonmerolae has been considered an excellent model system to understand the fundamental mechanisms behind the accumulation of TAG/starch in the microalga, as it has a smaller genome size and various biotechnological methods are available for it. Furthermore, C. merolae can grow and survive under high temperature (40 °C) and low pH (2-3) conditions, where most other organisms would die, thus making it a choice alga for large-scale production. Investigations using this alga has revealed that the target of rapamycin (TOR) kinase is involved in the accumulation of carbon-reserved molecules, TAGs, and starch. Furthermore, detailed molecular mechanisms of the role of TOR in controlling the accumulation of TAGs and starch were uncovered via omics analyses. Based on these findings, genetic engineering of the key gene and proteins resulted in a drastic increment of the amount of TAGs and starch. In addition to these studies, other trials that attempted to achieve the TAG increment in C. merolae have been summarized in this article.

Identification of Transcription Factors and the Regulatory Genes Involved in Triacylglycerol Accumulation in the Unicellular Red Alga Cyanidioschyzon merolae.

Microalgal triacylglycerols (TAGs) are a good feedstock for liquid biofuel production. Improving the expression and/or function of transcription factors (TFs) involved in TAG accumulation may increase TAG content; however, information on microalgae is still lacking. In this study, 14 TFs in the unicellular red alga Cyanidioschyzon merolae were identified as candidate TFs regulating TAG accumulation using available transcriptome and phosphoproteome data under conditions driving TAG accumulation. To investigate the roles of these TFs, we constructed TF-overexpression strains and analyzed lipid droplet (LD) formation and TAG contents in the cells grown under standard conditions. Based on the results, we identified four TFs involved in LD and TAG accumulation. RNA-Seq analyses were performed to identify genes regulated by the four TFs using each overexpression strain. Among the TAG biosynthesis-related genes, only the gene encoding the endoplasmic reticulum-localized lysophosphatidic acid acyltransferase 1 (LPAT1) was notably increased among the overexpression strains. In the LPAT1 overexpression strain, TAG accumulation was significantly increased compared with the control strain under normal growth conditions. These results indicate that the four TFs positively regulate TAG accumulation by changing their target gene expression in C. merolae.