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The red yeast Rhodosporidium toruloide is a versatile host for production of lipids and carotenoids. Genetic tools are underdeveloped for red yeasts due to their unique genetics and physiology. Currently expression of a heterogonous gene in red yeasts is largely based on integration of the designed cassette by Agrobacterium mediated transformation, yet this method is somewhat restricted when multiple genes are required to be expressed due to the lack of functional genetic elements. Here we demonstrate that virus 2A sequence is effective to mediate co-expression of multiple enzymes in R. toruloides. Two different 2A sequences, Porcine teschovirus-1 2A (P2A) and foot-and-mouth disease virus 2A (F2A), were evaluated. It was found that P2A sequence was more effective for co-expression of two antibiotic selection markers. Co-expression of three antibiotic resistance proteins was successful from a single promoter mediated by P2A sequence. When three heterogeneous enzymes responsible for β-carotene biosynthesis were co-expressed, recombinant R. toruloides strains produced up to 4.5-fold more β-carotene than that of the parental one. The use of 2A sequence can facilitate cassette construction to engineer advanced cell factories for production of lipids and related oleochemicals.

The Yunnan pine and Simao pine caterpillar moths, Dendrolimus houi Lajonquière and Dendrolimus kikuchii Matsumura (Lepidoptera: Lasiocampidae), are two closely related and sympatric pests of coniferous forests in southwestern China, and olfactory communication systems of these two insects have received considerable attention because of their economic importance. However, there is little information on the molecular aspect of odor detection about these insects. Furthermore, although lepidopteran species have been widely used in studies of insect olfaction, few work made comparison between sister moths on the olfactory recognition mechanisms. In this study, next-generation sequencing of the antennal transcriptome of these two moths were performed to identify the major olfactory genes. After comparing the antennal transcriptome of these two moths, we found that they exhibit highly similar transcripts-associated GO terms. Chemosensory gene families were further analyzed in both species. We identified 23 putative odorant binding proteins (OBP), 17 chemosensory proteins (CSP), two sensory neuron membrane proteins (SNMP), 33 odorant receptors (OR), and 10 ionotropic receptors (IR) in D. houi; and 27 putative OBPs, 17 CSPs, two SNMPs, 33 ORs, and nine IRs in D. kikuchii. All these transcripts were full-length or almost full-length. The predicted protein sequences were compared with orthologs in other species of Lepidoptera and model insects, including Bombyx mori, Manduca sexta, Heliothis virescens, Danaus plexippus, Sesamia inferens, Cydia pomonella, and Drosophila melanogaster. The sequence homologies of the orthologous genes in D. houi and D. kikuchii are very high. Furthermore, the olfactory genes were classed according to their expression level, and the highly expressed genes are our target for further function investigation. Interestingly, many highly expressed genes are ortholog gene of D. houi and D. kikuchii. We also found that the Classic OBPs were further separated into three groups according to their motifs, which will help future functional researches. Surprisingly, no pheromone receptor was identified in the two Dendrolimus species, which may indicate a special pheromone identification mechanism in Dendrolimus. Our work allows for further functional studies of pheromones and host volatile recognition genes, and give novel candidate targets for pest management., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The Masculinizer gene, Masc, encodes a lepidopteran‐specific novel CCCH‐type zinc finger protein, which controls sex determination and dosage compensation in Bombyx mori. Considering the potential application of it in pest control, it is necessary to investigate the function of Masc gene in Hyphantria cunea, a globally invasive forest pest. In the present study, we identified and functionally characterized the Masc gene, HcMasc, in H. cunea. Sequence analysis revealed that HcMASC contained the conserved CCCH‐type zinc finger domain, nuclear localization signal, and male determining domain, in which the last was confirmed to be required for its masculinization in BmN cell line. However, expression data showed that unlike male‐biased expression in B. mori, HcMasc gene expresses in main all developmental stages or tissues in both sexes. Clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR‐associated protein 9‐based disruption of the common exons 1 and 3 of the HcMasc gene resulted in imbalanced sex ratio and abnormal external genitalia of both sexes. Our results suggest that the HcMasc gene is required for both male and female sexual differentiation and dosage compensation in H. cunea and provide a foundation for developing better strategies to control this pest. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a widely used technique for gene function researches and recently pest controls. It had been applied in emerald ash borer (EAB Agrilus planipennis) larvae and adults, and achieved significant interference effects, whether by ingesting or microinjecting. Feeding in the phloem and cambial regions, the larvae of A. planipennis are difficult to be controlled by conventional insecticides, so adult stage is the critical stage for EAB control. However, the target genes of adult stage of A. planipennis need to be further screened. Here, we preliminarily screened five potential target genes of vision and olfaction for RNAi in A. planipennis. Three odorant binding proteins (OBPs) and three opsins, which expressed significantly different between newly emerged and sexually mature EABs (OBP5, OBP7, OBP10, LW opsin 1 and UV opsin 2) or highly in sexually mature male EAB (UV opsin 3), were selected as targets to design primers for gene silencing. After dsRNA injection, the gene expression levels were determined by real-time quantitative PCR. We found that the expression levels of five genes were significantly down-regulated, during the 4 days after dsRNA injection. Among these genes, the expression of LW opsin 1 was down-regulated the most, causing a reduction of 99.1% compared with the control treated with EGFP dsRNA, followed by UV opsin 3 (97.4%), UV opsin 2 (97.0%), OBP7 (96.2%), and OBP10 (88.7%). This study provides a basis for further RNAi-based new controlling method development of A. planipennis at adult stage. [ABSTRACT FROM AUTHOR]

Highlights • p12-knockout generates a HR deficiency phenotype. • Pol δ 4 is required for full expression of cellular HR activity. • p12 knockout cells are sensitized to PARP inhibitors. • p12 expression is a potential biomarker for the use of PARP inhibitors. • p12 deficiency may have a role in SCLC tumorigenesis. Abstract Human DNA polymerase δ is normally present in unstressed, non-dividing cells as a heterotetramer (Pol δ 4). Its smallest subunit, p12, is transiently degraded in response to UV damage, as well as during the entry into S-phase, resulting in the conversion of Pol δ 4 to a trimer (Pol δ 3). In order to further understand the specific cellular roles of these two forms of Pol δ, the gene (POLD4) encoding p12 was disrupted by CRISPR/Cas9 to produce p12 knockout (p12KO) cells. Thus, Pol δ 4 is absent in p12KO cells, leaving Pol δ 3 as the sole source of Pol δ activity. GFP reporter assays revealed that the p12KO cells exhibited a defect in homologous recombination (HR) repair, indicating that Pol δ4, but not Pol δ 3, is required for HR. Expression of Flag-tagged p12 in p12KO cells to restore Pol δ 4 alleviated the HR defect. These results establish a specific requirement for Pol δ 4 in HR repair. This leads to the prediction that p12KO cells should be more sensitive to chemotherapeutic agents, and should exhibit synthetic lethal killing by PARP inhibitors. These predictions were confirmed by clonogenic cell survival assays of p12KO cells treated with cisplatin and mitomycin C, and with the PARP inhibitors Olaparib, Talazoparib, Rucaparib, and Niraparib. The sensitivity to PARP inhibitors in H1299-p12KO cells was alleviated by expression of Flag-p12. These findings have clinical significance, as the expression levels of p12 could be a predictive biomarker of tumor response to PARP inhibitors. In addition, small cell lung cancers (SCLC) are known to exhibit a defect in p12 expression. Analysis of several SCLC cell lines showed that they exhibit hypersensitivity to PARP inhibitors, providing evidence that loss of p12 expression could represent a novel molecular basis for HR deficiency. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the expression of FGF2 and FGFR2 in patients with idiopathic pulmonary fibrosis (IPF) and lung cancer (LC) as well as their clinical significance. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting were used to detect FGF2 and FGFR2 expression in LC and adjacent normal tissues of LC patients and lavage fluid of idiopathic pulmonary fibers patients and normal controls (confirmed by bronchoalveolar lavage examination). The expression levels of FGF2 mRNA and protein in the non-small cell LC tissues were significantly higher than those in the adjacent normal tissues (P<0.001). The expression level of FGF2 protein in lavage fluid of patients with IPF was higher than that of the control group (P<0.001). The expression level of FGFR2 mRNA in the non-small cell LC tissues was significantly higher than that in the adjacent normal tissues (P<0.001). The expression level of FGFR2 protein in the non-small cell LC tissues was higher than that in the adja- adjacent normal lung tissues (P<0.001). The expression levels of FGF2 mRNA and FGFR2 mRNA in cancer tissues were not significantly correlated with age, sex and history of smoking (P>0.05), but were significantly correlated with lymph node metastasis, tumor differentiation and TNM staging. FGF2 and FGFR2 proteins were highly expressed in cancer tissues of LC patients and lavage fluid of patients with IPF. The expression of FGF2 mRNA and FGFR2 mRNA was correlated with lymph node metastasis and TNM stage. The high expression levels of FGF2 mRNA and FGFR2 mRNA were associated with tumor metastasis and poor prognosis of LC patients. [ABSTRACT FROM AUTHOR]

Pheromone-binding proteins (PBPs) play an important role in the recognition of pheromones by insects. However, the abilities of these PBPs to discriminate pheromone components and recognize the isomers are unclear. Dendrolimus houi and Dendrolimus kikuchii are two sympatric coniferous pests whose pheromones have cis-trans isomers. We used these insect species to detect the precise recognition abilities of PBPs. The four PBPs examined showed male-biased antenna-intensive expression patterns, whereas PBP1 showed higher expression than PBP2 in the antenna. DhouPBP1 only bound to a minor interspecific pheromone component, whereas DhouPBP2 bound to all three intraspecific components and another minor interspecific component. DkikPBP1 and DkikPBP2 could recognize all three intraspecific components with affinities negatively correlated with their ratios, and they bound to interspecific pheromones with affinity that was positively correlated with the ratios. The four PBPs have different cis-trans isomer discrimination abilities, i.e., DhouPBP1 and DkikPBP1 could not discriminate the two cis-trans isomer pairs of pheromones from the two species, whereas DhouPBP2 could discriminate between both pairs, and DkikPBP2 could only discriminate one pair. Overall, PBPs from D. houi and D. kikuchii use different strategies to help the moths to discriminate the intra- and interspecific pheromone components. Our work will contribute to better understanding of the sex pheromone recognition mechanism in these two sister species of moths and provide insights into more effective management practices of these pest species. [ABSTRACT FROM AUTHOR]

Odorant binding proteins (OBPs) play important roles in insect olfactory processes. The Chinese pine caterpillar moth, Dendrolimus tabulaeformis (Lepidoptera, Lasiocampidae) is a serious economic pest in China, and the pheromones of this species have been identified to monitor their presence. However, the molecular mechanisms by which D. tabulaeformis perceive pheromones and host volatiles remain unknown. In this study, we identified and characterized three new OBPs, including one pheromone binding protein (PBP1) and two general odor binding proteins (GOBPs), from antennal cDNA of D. tabulaeformis. The deduced amino acid sequences of DtabPBP1, DtabGOBP1, and DtabGOBP2 revealed mature proteins of 140, 147, and 140 amino acids, respectively. Each has six cysteine residues in conserved positions relative to other known OBPs. Amino-acid alignments indicated that the two GOBPs are more conserved (DtabGOBP1 is 52.9-67.4 % identical to orthologs from other Lepidoptera, and DtabGOBP2 is 55.2-81.8 % identical) than the PBP (32.5-46.0 %). Real-time PCR indicated tissue- and sex-specific expression patterns of the three genes. DtabPBP1 was mainly expressed in the antennae of males, whereas female antennae had only 1.09 % the expression in male antennae. Both DtabGOBP1 and DtabGOBP2 were more highly expressed in antennae than in other tissues, while DtabGOBP1 was more abundant in male antennae and DtabGOBP2 in female antennae. In addition, the binding specificities of the three proteins were investigated, and all three OBPs exhibited high binding affinities for the pheromone component (5 Z,7 E)-5,7-dodecadien-1-yl propionate ( Z5, E7-12:OPr). This suggests a role in binding pheromone for GOBPs, as well as PBP1, in D. tabulaeformis. [ABSTRACT FROM AUTHOR]

The pine wood nematode (PWN) Bursaphelenchus xylophilus has caused disastrous losses of pine forests in many countries, and the success of PWN depends strongly on interactions with its insect vectors. Monochamus saltuarius is a newly recorded vector in Northeast China. Feeding (i.e., immature) and egg-laying (i.e., mature) Monochamus spp. target different host plants, and olfactory cues play important roles regarding host choice. Whether infestation with PWN affects olfactory mechanisms in M. saltuarius related to feeding and oviposition is of interest as this may affect the spread of nematodes to new healthy hosts. However, little is known about molecular mechanisms of the olfactory system of M. saltuarius. We identified chemosensory-related genes in adult M. saltuarius and examined the influence of B. xylophilus on the respective expression patterns. Fifty-three odorant-binding proteins (OBPs), 15 chemosensory proteins, 15 olfactory receptors (ORs), 10 gustatory receptors, 22 ionotropic receptors (IRs), and two sensory neuron membrane proteins were identified, and sex bias among non-infested beetles was mainly found with respect to expression of OBPs. Interestingly, OBPs and ORs were markedly down-regulated in male M. saltuarius infested with B. xylophilus, which may reduce olfactory sensitivity of male M. saltuarius and affect the spreading of B. xylophilus to new hosts. Our results will help understand the interactions between B. xylophilus and M. saltuarius, which may lead to the identification of new control targets in the olfactory system of M. saltuarius. [ABSTRACT FROM AUTHOR]

Early biochemical studies have demonstrated that lipid accumulation by oleaginous yeasts is linked to the activity of the NAD-dependent isocitrate dehydrogenase (Idh). However, molecular study of Idh of oleaginous microorganisms remains limited. Here, we present the cloning of a mitochondrial NAD-specific Idh from Rhodosporidium toruloides (RtIdh), an excellent microbial lipid producer that uses carbohydrates as the carbon source. The evolutionary relationship analyses among RtIdhs and other yeast Idhs revealed that RtIdh had a closer relationship with the Idhs of Ustilago maydis and Schizophyllum commune. We expressed the Rt IDH gene in the yeast Saccharomyces cerevisiae idhΔ mutant. Under the nitrogen-limited condition, the intracellular lipid content and extracellular citrate concentration of the culture of the S. cerevisiae idhΔ carrying the Rt IDH gene increased as the carbon/nitrogen molar ratio of the media increased, while the wild-type S. cerevisiae strain showed no correlation. Our data provided valuable information for elucidating the molecular mechanism of microbial oleaginicity and for engineering microorganisms to produce metabolites of fatty acid pathway. [ABSTRACT FROM AUTHOR]

Abstract: The glycoside hydrolase β-1,3-glucomannanase is an enzyme that specifically breaks the β-1,3 glycosidic bond of the glucomannan, the main cell wall constituent of some yeasts. In this work, a codon optimized DNA sequence of the MAN5C gene from Penicillium lilacinum ATCC 36010 was expressed in the yeast Pichia pastoris under the control of AOX1 promoter. The recombinant protein plMAN5C was purified from the shake flask culture and the stirred-tank bioreactor culture in yields of 30.0mg/l and 224.0mg/l, respectively. The purified protein had a specific activity of 14.6U/mg at 37°C, pH 4.5. Biochemical analysis showed that the optimal temperature and pH for plMAN5C were 50°C and 4.5, respectively. The recombinant plMAN5C was efficient in lysis of the cell wall of the red yeast Rhodosporidium toruloides to form protoplast. Our work provided an effective system for heterogeneous production of β-1,3-glucomannanase, which should facilitate a more convenient application of this enzyme in biotechnology and other related areas. [Copyright &y& Elsevier]

Worldwide, pine forests have been threatened by a devastating pine wood disease caused by Bursaphelenchus xylophilus, with Monochamus saltuarius being a newly recorded vector of the disease in Northeast China. The olfactory system plays important roles in both feeding and oviposition during the adult stage of M. saltuarius, and olfactory gene function research is essential for gaining an understanding of the olfactory mechanisms of this pest. However, there is limited information available regarding olfactory gene functions in this pest. In the present study, we selected 7 olfactory genes, including 2 chemosensory proteins, 2 odorant‐binding proteins, the odorant co‐receptor and 2 odorant receptors, which were relatively highly expressed during the adult stage. We silenced these genes by RNA interference (RNAi), and real‐time quantitative PCR was used to detect their expression levels after double‐stranded RNA (dsRNA) injection. The results indicate that these genes were significantly downregulated at 2 d post‐dsRNA injection, and this was sustained until 5 d post‐injection. Electroantennography tests indicated that the knockdown of MsalOBP14 and MsalOrco impaired the olfactory response of M. saltuarius to 11 host volatiles and 1 sex pheromone compound. Y‐tube experiments further confirmed that downregulated MsalOBP14 and MsalOrco expression led to olfactory dysfunction in M. saltuarius, which significantly lost selectivity. The results indicate that MsalOBP14 and MsalOrco play critical roles in sex communication and host volatile detection in M. saltuarius, and possibly represent 2 effective targets for controlling this forest pest through olfactory disruption. [ABSTRACT FROM AUTHOR]

The red yeast Rhodosporidium toruloides is an excellent microbial host for production of carotenoids, neutral lipids and valuable enzymes. In recent years, genetic tools for gene expression and gene disruption have been developed for this red yeast. However, methods remain limited in terms of fine-tuning gene expression. In this study, we first demonstrated successful implementation of RNA interference (RNAi) in R. toruloides NP11, which was applied to down-regulate the expression of autophagy related gene 8 (ATG8), and fatty acid synthase genes (FAS1 and FAS2), respectively. Compared with the control strain, RNAi-engineered strains showed a silencing efficiency ranging from 11% to 92%. The RNAi approach described here ensures selective inhibition of the target gene expression, and should expand our capacity in the genetic manipulation of R. toruloides for both fundamental research and advanced cell factory development. [ABSTRACT FROM AUTHOR]

Abstract: Metabolomics is a potent tool to assist in identifying the function of unknown genes through analysis of metabolite changes in the context of varied genetic backgrounds. However, the availability of a universal unbiased profiling analysis is still a big challenge. In this study, we report an optimized metabolic profiling method based on gas chromatography–mass spectrometry for Escherichia coli. It was found that physiological saline at −80°C could ensure satisfied metabolic quenching with less metabolite leakage. A solution of methanol/water (21:79, v/v) was proved to be efficient for intracellular metabolite extraction. This method was applied to investigate the metabolome difference among wild-type E. coli, its yfcC deletion, and overexpression mutants. Statistical and bioinformatic analysis of the metabolic profiling data indicated that the expression of yfcC potentially affected the metabolism of glyoxylate shunt. This finding was further validated by real-time quantitative polymerase chain reactions showing that expression of aceA and aceB, the key genes in glyoxylate shunt, was upregulated by yfcC. This study exemplifies the robustness of the proposed metabolic profiling analysis strategy and its potential roles in investigating unknown gene functions in view of metabolome difference. [Copyright &y& Elsevier]