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.

Disruption of SMC-related genes promotes recombinant cholesterol esterase production in Burkholderia stabilis.

Burkholderia stabilis strain FERMP-21014 secretes cholesterol esterase (BsChe), which is used in clinical settings to determine serum cholesterol levels. Previously, we constructed an expression plasmid with an endogenous constitutive promoter to enable the production of recombinant BsChe. In this study, we obtained one mutant strain with 13.1-fold higher BsChe activity than the wild type, using N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen. DNA-sequencing analysis revealed that the strain had lost chromosome 3 (∆Chr3), suggesting that the genes hindering BsChe production may be encoded on Chr3. We also identified common mutations in the functionally unknown BSFP_068720/30 genes in the top 10 active strains generated during transposon mutagenesis. As BSFP_068720/30/40 comprised an operon on Chr3, we created the BSFP_068720/30/40 disruption mutant and confirmed that each disruption mutant containing the expression plasmid exhibited ~ 16.1-fold higher BsChe activity than the wild type. Quantitative PCR showed that each disruption mutant and ΔChr3 had a ~ 9.4-fold higher plasmid copy number than the wild type. Structural prediction models indicate that BSFP_068730/40 is structurally homologous to the structural maintenance of chromosomes (SMC) protein MukBE, which is responsible for chromosome segregation during cell division. Conversely, BSFP_068720/30/40 disruption did not lead to a Chr3 drop-out. These results imply that BSFP_068720/30/40 is not a SMC protein but is involved in destabilizing foreign plasmids to prevent the influx of genetic information from the environment. In conclusion, the disruption of BSFP_068720/30/40 improved plasmid stability and copy number, resulting in exceptionally high BsChe production. KEY POINTS: • Disruption of BSFP_068720/30/40 enabled mass production of Burkholderia Che/Lip. • BSFP_068730/40 is an SMC protein homolog not involved in chromosome retention. • BSFP_068720/30/40 is likely responsible for the exclusion of exogenous plasmids.

Multivariate statistical monitoring system for microbial population dynamics.

Microbiomes in their natural environments vary dynamically with changing environmental conditions. The detection of these dynamic changes in microbial populations is critical for understanding the impact of environmental changes on the microbial community. Here, we propose a novel method to detect time-series changes in the microbiome, based on multivariate statistical process control. By focusing on the interspecies structures, this approach enables the robust detection of time-series changes in a microbiome composed of a large number of microbial species. Applying this approach to empirical human gut microbiome data, we accurately traced time-series changes in microbiota composition induced by a dietary intervention trial. This method was also excellent for tracking the recovery process after the intervention. Our approach can be useful for monitoring dynamic changes in complex microbial communities.

Selection of the optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli.

We have constructed an Escherichia coli-based platform producing (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids (BIAs), using up to 14 genes. (S)-reticuline was produced from a simple carbon source such as glucose and glycerol via L-DOPA, which is synthesized by hydroxylation of L-tyrosine, one of the rate-limiting steps of the reaction. There are three kinds of enzymes catalyzing tyrosine hydroxylation: tyrosinase (TYR), tyrosine hydroxylase (TH), and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, to further improve (S)-reticuline production, we chose eight from these three kinds of tyrosine hydroxylation enzymes (two TYRs, four THs, and two HpaBCs) derived from various organisms, and examined which enzyme was optimal for (S)-reticuline production in E. coli. TH from Drosophila melanogaster was the most suitable for (S)-reticuline production under the experimental conditions tested. We improved the productivity by genome integration of a gene set for L-tyrosine overproduction, introducing the regeneration pathway of BH4, a cofactor of TH, and methionine addition to enhance the S-adenosylmethionine supply. As a result, the yield of (S)-reticuline reached up to 384 µM from glucose in laboratory-scale shake flask. Furthermore, we found three inconsistent phenomena: an inhibitory effect due to additional gene expression, conflicts among the experimental conditions, and interference of an upstream enzyme from an additional downstream enzyme. Based on these results, we discuss future perspectives and challenges of integrating multiple enzyme genes for material production using microbes. Graphical abstract The optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli KEY POINTS: • There are three types of enzymes catalyzing tyrosine hydroxylation reaction: tyrosinase, tyrosine hydroxylase, and 4-hydroxyphenylacetate 3-monooxygenase. • Tyrosine hydroxylase from Drosophila melanogaster exhibited the highest activity and was suitable for (S)-reticuline production in E. coli. • New insights were provided on constructing an alkaloid production system with multi-step reactions in E. coli.

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.

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.

Production of recombinant extracellular cholesterol esterase using consistently active promoters in Burkholderia stabilis.

Burkholderia stabilis FERMP-21014 produces highly active cholesterol esterase in the presence of fatty acids. To develop an overexpression system for cholesterol esterase production, we carried out RNA sequencing analyses to screen strongly active promoters in FERMP-21014. Based on gene expression consistency analysis, we selected nine genes that were consistently expressed at high levels, following which we constructed expression vectors using their promoter sequences and achieved overproduction of extracellular cholesterol esterase under fatty acid-free conditions. Of the tested promoters, the promoter of BSFP_0720, which encodes the alkyl hydroperoxide reductase subunit AhpC, resulted in the highest cholesterol esterase activity (24.3 U mL-1). This activity level was 243-fold higher than that of the wild-type strain under fatty acid-free conditions. We confirmed that cholesterol esterase was secreted without excessive accumulation within the cells. The gene expression consistency analysis will be useful to screen promoters applicable to the overexpression of other industrially important enzymes.

Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome.

Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (ß-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

Prediction of developmental chemical toxicity based on gene networks of human embryonic stem cells.

Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here, we show that toxicity category prediction by support vector machines (SVMs), which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system, is improved by the adoption of gene networks, in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5-100% for three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals, bisphenol-A and permethrin, our system yielded reasonable results: bisphenol-A was categorized as an NGC, and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system, it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs, our study has considerable potential to predict late-onset chemical toxicities, including abnormalities that occur during embryonic development.

Gene expression profiling of hepatitis B- and hepatitis C-related hepatocellular carcinoma using graphical Gaussian modeling.

BACKGROUND & AIMS: Gene expression profiling of hepatocellular carcinoma (HCC) and background liver has been studied extensively; however, the relationship between the gene expression profiles of different lesions has not been assessed. METHODS: We examined the expression profiles of 34 HCC specimens (17 hepatitis B virus [HBV]-related and 17 hepatitis C virus [HCV]-related) and 71 non-tumor liver specimens (36 chronic hepatitis B [CH-B] and 35 chronic hepatitis C [CH-C]) using an in-house cDNA microarray consisting of liver-predominant genes. Graphical Gaussian modeling (GGM) was applied to elucidate the interactions of gene clusters among the HCC and non-tumor lesions. RESULTS: In CH-B-related HCC, the expression of vascular endothelial growth factor-family signaling and regulation of T cell differentiation, apoptosis, and survival, as well as development-related genes was up-regulated. In CH-C-related HCC, the expression of ectodermal development and cell proliferation, wnt receptor signaling, cell adhesion, and defense response genes was also up-regulated. Many of the metabolism-related genes were down-regulated in both CH-B- and CH-C-related HCC. GGM analysis of the HCC and non-tumor lesions revealed that DNA damage response genes were associated with AP1 signaling in non-tumor lesions, which mediates the expression of many genes in CH-B-related HCC. In contrast, signal transducer and activator of transcription 1 and phosphatase and tensin homolog were associated with early growth response protein 1 signaling in non-tumor lesions, which potentially promotes angiogenesis, fibrogenesis, and tumorigenesis in CH-C-related HCC. CONCLUSIONS: Gene expression profiling of HCC and non-tumor lesions revealed the predisposing changes of gene expression in HCC. This approach has potential for the early diagnosis and possible prevention of HCC.

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.

Social isolation shortens lifespan through oxidative stress in ants.

Social isolation negatively affects health, induces detrimental behaviors, and shortens lifespan in social species. Little is known about the mechanisms underpinning these effects because model species are typically short-lived and non-social. Using colonies of the carpenter ant Camponotus fellah, we show that social isolation induces hyperactivity, alters space-use, and reduces lifespan via changes in the expression of genes with key roles in oxidation-reduction and an associated accumulation of reactive oxygen species. These physiological effects are localized to the fat body and oenocytes, which perform liver-like functions in insects. We use pharmacological manipulations to demonstrate that the oxidation-reduction pathway causally underpins the detrimental effects of social isolation on behavior and lifespan. These findings have important implications for our understanding of how social isolation affects behavior and lifespan in general.

Improvement of Free Fatty Acid Secretory Productivity in Aspergillus oryzae by Comprehensive Analysis on Time-Series Gene Expression.

Aspergillus oryzae is a filamentous fungus that has historically been utilized in the fermentation of food products. In recent times, it has also been introduced as a component in the industrial biosynthesis of consumable compounds, including free fatty acids (FFAs), which are valuable and versatile products that can be utilized as feedstocks in the production of other commodities, such as pharmaceuticals and dietary supplements. To improve the FFA secretory productivity of A. oryzae in the presence of Triton X-100, we analyzed the gene expression of a wild-type control strain and a disruptant strain of an acyl-CoA synthetase gene, faaA, in a time-series experiment. We employed a comprehensive analysis strategy using the baySeq, DESeq2, and edgeR algorithms to clarify the vital pathways for FFA secretory productivity and select genes for gene modification. We found that the transport and metabolism of inorganic ions are crucial in the initial stages of FFA production and revealed 16 candidate genes to be modified in conjunction with the faaA disruption. These genes were verified through the construction of overexpression strains, and showed that the manipulation of reactions closer to the FFA biosynthesis step led to a higher increase in FFA secretory productivity. This resulted in the most successful overexpression strains to have an FFA secretory productivity more than two folds higher than that of the original faaA disruptant. Our study provides guidance for further gene modification for FFA biosynthesis in A. oryzae and for enhancing the productivity of other metabolites in other microorganisms through metabolic engineering.

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.

Bacterial triacylglycerol lipase is a potential cholesterol esterase: Identification of a key determinant for sterol-binding specificity.

Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

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.

Rapid reprogramming of tumour cells into cancer stem cells on double-network hydrogels.

Cancer recurrence can arise owing to rare circulating cancer stem cells (CSCs) that are resistant to chemotherapies and radiotherapies. Here, we show that a double-network hydrogel can rapidly reprogramme differentiated cancer cells into CSCs. Spheroids expressing elevated levels of the stemness genes Sox2, Oct3/4 and Nanog formed within 24 h of seeding the gel with cells from any of six human cancer cell lines or with brain cancer cells resected from patients with glioblastoma. Human brain cancer cells cultured on the double-network hydrogel and intracranially injected in immunodeficient mice led to higher tumorigenicity than brain cancer cells cultured on single-network gels. We also show that the double-network gel induced the phosphorylation of tyrosine kinases, that gel-induced CSCs from primary brain cancer cells were eradicated by an inhibitor of the platelet-derived growth factor receptor, and that calcium channel receptors and the protein osteopontin were essential for the regulation of gel-mediated induction of stemness in brain cancer cells.

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.

Genome Sequence of Rhodococcus erythropolis Type Strain JCM 3201.

Rhodococcus erythropolis JCM 3201 can express several recombinant proteins that are difficult to express in Escherichia coli It is used as one of the hosts for protein expression and bioconversion. Here, we report the draft genome sequence of R. erythropolis JCM 3201.