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Background: The polyene macrocyclic compound amphotericin B (AmB) is an important antifungal antibiotic for the clinical treatment of invasive fungal infections. To rationally guide the improvement of AmB production in the main producing strain Streptomyces nodosus, comparative metabolomics analysis was performed to investigate the intracellular metabolic changes in wild-type S. nodosus ZJB20140315 with low-yield AmB production and mutant S. nodosus ZJB2016050 with high-yield AmB production, the latter of which reached industrial criteria on a pilot scale. Results: To investigate the relationship of intracellular metabolites, 7758 metabolites were identified in mutant S. nodosus and wildtype S. nodosus via LC–MS. Through analysis of metabolism, the level of 26 key metabolites that involved in carbon metabolism, fatty acids metabolism, amino acids metabolism, purine metabolism, folate biosynthesis and one carbon pool by folate were much higher in mutant S. nodosus. The enrichment of relevant metabolic pathways by gene overexpression strategy confirmed that one carbon pool by folate was the key metabolic pathway. Meanwhile, a recombinant strain with gene metH (methionine synthase) overexpressed showed 5.03 g/L AmB production within 120 h fermentation, which is 26.4% higher than that of the mutant strain. Conclusions: These results demonstrated that comparative metabolomics analysis was an effective approach for the improvement of AmB production and could be applied for other industrially or clinically important compounds as well. [ABSTRACT FROM AUTHOR]

Background: Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12–C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts. Results: Schlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative ferredoxin genes in vivo and identified two ferredoxin reductases, FdR_0130 and FdR_7100, by whole-cell biotransformation of epothilone C to effectively support the catalytic activity of EpoK. In addition, we obtained strain H7029-3, with a high epothilone B yield and found that the proportion of epothilone A + B produced by this strain was 90.93%. Moreover, the whole-cell bioconversion strain 7029-10 was obtained; this strain exhibited an epothilone C conversion rate of 100% in 12 h. Further RT-qPCR experiments were performed to analyse the overexpression levels of the target genes. Gene knock-out experiments showed that the selected ferredoxin (Fdx_0135) and its reductases (FdR_0130 and FdR_7100) might participate in critical physiological processes in DSM 7029. Conclusion: Gene overexpression and whole-cell biotransformation were effective methods for identifying the electron transport partners of the P450 enzyme EpoK. In addition, we obtained an epothilone B high-yield strain and developed a robust whole-cell biotransformation system. This strain and system hold promise for the industrial production of epothilone B and its derivatives. [ABSTRACT FROM AUTHOR]

Background: Drought stress is one of the major factors limiting wheat production globally. Improving drought tolerance is important for agriculture sustainability. Although various morphological, physiological and biochemical responses associated with drought tolerance have been documented, the molecular mechanisms and regulatory genes that are needed to improve drought tolerance in crops require further investigation. We have used a novel 4-component version (for overexpression) and a 3-component version (for underexpression) of a barley stripe mosaic virus-based (BSMV) system for functional characterization of the C2H2-type zinc finger protein TaZFP1B in wheat. These expression systems avoid the need to produce transgenic plant lines and greatly speed up functional gene characterization. Results: We show that overexpression of TaZFP1B stimulates plant growth and up-regulates different oxidative stress-responsive genes under well-watered conditions. Plants that overexpress TaZFP1B are more drought tolerant at critical periods of the plant's life cycle. Furthermore, RNA-Seq analysis revealed that plants overexpressing TaZFP1B reprogram their transcriptome, resulting in physiological and physical modifications that help wheat to grow and survive under drought stress. In contrast, plants transformed to underexpress TaZFP1B are significantly less tolerant to drought and growth is negatively affected. Conclusions: This study clearly shows that the two versions of the BSMV system can be used for fast and efficient functional characterization of genes in crops. The extent of transcriptome reprogramming in plants that overexpress TaZFP1B indicates that the encoded transcription factor is a key regulator of drought tolerance in wheat. [ABSTRACT FROM AUTHOR]

Background: Glucoamylase is one of the most industrially applied enzymes, produced by Aspergillus species, like Aspergillus niger. Compared to the traditional ways of process optimization, the metabolic engineering strategies to improve glucoamylase production are relatively scarce. Results: In the previous study combined multi-omics integrative analysis and amino acid supplementation experiment, we predicted four amino acids (alanine, glutamate, glycine and aspartate) as the limited precursors for glucoamylase production in A. niger. To further verify this, five mutants namely OE-ala, OE-glu, OE-gly, OE-asp1 and OE-asp2, derived from the parental strain A. niger CBS 513.88, were constructed respectively for the overexpression of five genes responsible for the biosynthesis of the four kinds of amino acids (An11g02620, An04g00990, An05g00410, An04g06380 and An16g05570). Real-time quantitative PCR revealed that all these genes were successfully overexpressed at the mRNA level while the five mutants exhibited different performance in glucoamylase production in shake flask cultivation. Notably, the results demonstrated that mutant OE-asp2 which was constructed for reinforcing cytosolic aspartate synthetic pathway, exhibited significantly increased glucoamylase activity by 23.5% and 60.3% compared to CBS 513.88 in the cultivation of shake flask and the 5 L fermentor, respectively. Compared to A. niger CBS 513.88, mutant OE-asp2 has a higher intracellular amino acid pool, in particular, alanine, leucine, glycine and glutamine, while the pool of glutamate was decreased. Conclusion: Our study combines the target prediction from multi-omics analysis with the experimental validation and proves the possibility of increasing glucoamylase production by enhancing limited amino acid biosynthesis. In short, this systematically conducted study will surely deepen the understanding of resources allocation in cell factory and provide new strategies for the rational design of enzyme production strains. [ABSTRACT FROM AUTHOR]

Background: Polysaccharides and poly-γ-glutamic acid (γ-PGA) are biomacromolecules that have been reported as bioflocculants, and they exhibit high flocculating activity in many industrial applications. Bacillus licheniformis CGMCC 2876 can produce polysaccharide and γ-PGA bioflocculants under different culture conditions. Several key genes are involved in the metabolic pathway of polysaccharides in B. licheniformis, but the impacts of the regulation of these genes on the production of polysaccharide bioflocculants have not been illustrated completely. To increase the bioflocculant production and identify the correlation between the synthesis of polysaccharides and γ-PGA in B. licheniformis, a few key genes were investigated to explore their influence on the synthesis of the bioflocculants. Results: Overexpressing epsB from the eps gene cluster not only improved the bioflocculant crude yield by 13.98% but also enhanced the flocculating activity by 117.92%. The composition of the bioflocculant from the epsB recombinant strain was 28.95% total sugar, 3.464% protein and 44.03% γ-PGA, while in the original strain, these components represented 53.67%, 3.246% and 34.13%, respectively. In combination with an analysis of the transcriptional levels of several key genes involved in γ-PGA synthesis in B. licheniformis, we inferred that epsB played a key role in the synthesis of both polysaccharide and γ-PGA. The bioflocculant production of the epsB recombinant strain was further evaluated during batch fermentation in a 2 L fermenter; the flocculating activity reached 9612.75 U/mL, and the bioflocculant yield reached 10.26 g/L after 72 h, representing increases of 224% and 36.62%, respectively, compared with the original strain. Moreover, we found that the tandem expression of phosphoglucomutase (pgcA) and UTP-glucose- 1-phosphate uridylyltransferase (gtaB1) could enhance the crude yield of the bioflocculant by 20.77% and that the overexpression of epsA could enhance the bioflocculant yield by 23.70% compared with the original strain. Conclusions: This study provides a new method to greatly increase the bioflocculant production in B. licheniformis, and it demonstrates the correlation between the biosynthesis of polysaccharide and γ-PGA during EPS fermentation by regulating the expression of EpsB. [ABSTRACT FROM AUTHOR]

Background: The barley stripe mosaic virus (BSMV) has become a popular vector to study gene function in cereals. However, studies have been limited to gene silencing in leaves of barley or wheat. In addition, the method produces high variability between different leaves and plants. To overcome these limitations, we explored the potential of modifying the inoculation protocol for BSMV gene overexpression. An improved light, oxygen or voltage-sensing (iLOV) domain-based fluorescent protein was used as a reporter of gene expression to monitor the infection and spread of BSMV. Tobacco (Nicotiana benthamiana) leaves were infected via agroinfiltration and the leaves were homogenized to extract the BSMV particles and inoculate wheat tissues using the traditional leaf abrasion method or by incubation during seed imbibition in a Petri dish. Results: Compared to the leaf abrasion method, the seed imbibition method resulted in a high and uniform detection of iLOV in both roots and leaves of different wheat cultivars and other monocot and dicot species within 7 days after germination. The progression of viral infection via the imbibition method as measured by the expression of iLOV was more stable in different organs and tissues and is transmissible to the next generation. Conclusion: Our results show that BSMV can be used as a vector for the expression of small genes such as iLOV in wheat roots and leaves. The inoculation by seed imbibition allows genes to be expressed rapidly and uniformly in wheat and different monocot and dicot species compared to the traditional leaf abrasion method. It also produces high successful transformation as early as 7 days post infection allowing gene function studies during the first generation of infected plants. Furthermore, the method is simple, rapid, and inexpensive compared to the production of transgenic plants. [ABSTRACT FROM AUTHOR]

Background Ovarian carcinoma is one of the most common gynecological cancers with high mortality rates. Numerous evidences demonstrate that cancer cells undergo metabolic abnormality during tumorigenesis in tumor microenvironment and further facilitate tumor progression. Succinate dehydrogenase (SDH or Complex II) is one of the important enzymes in the tricarboxylic acid (TCA) cycle. Succinate dehydrogenase subunit B (SDHB) gene, which encodes one of the four subunits of SDH, has been recognized as a tumor suppressor. However the role of SDHB in ovarian cancer is still unclear. Methods Using the SDHB specific siRNA and overexpression plasmid, the expression of SDHB was silenced and conversely induced in ovarian cancer cell lines SKOV3 and A2780, respectively. The possible role of SDHB in ovarian cancer was investigated in vitro, using proliferation, migration and invasion assays. To explore the mechanism, proliferation and migration related proteins such as Bcl-2, cleaved caspase 3, p-ERK, MMP-2, and p-FAK were examined by western blot. P-P38, p-AMPKα, and HIF-1α were also examined by western blot. CoCl2 was used to induce HIF-1α expression in SKOV3 and A2780 cells. Results SDHB silencing promoted cell proliferation, invasion, and migration, but inhibited apoptosis of SKOV3 and A2780 cells. In contrast, overexpression of SDHB inhibited cell proliferation, invasion, migration, and promoted apoptosis in SKOV3 cells. It was observed that up-regulation of Bcl-2 and MMP-2, activation of p-P38, p-ERK, and p-FAK, inhibition of cleaved caspase 3 in SDHB-silenced cells. Meanwhile, decreased Bcl-2 and MMP-2, inhibition of p-P38, p-ERK, and p-FAK, activation of cleaved caspase 3 were shown in SDHB-overexpressed SKOV3 cells. HIF-1α, an essential factor in tumor progression, was up-regulated in SDHB-silenced cells with the activation of p-AMPKα and down-regulated in SDHB-overexpressed cancer cells with the decreased p-AMPKα. And SDHB was proved to be decreased due to upregulation of HIF-1α expression in CoCl2-treated cancer cells. Conclusions Our results firstly revealed that SDHB played a key role in cell proliferation, invasion, migration, and apoptosis of human ovarian carcinoma via AMPK-HIF-1α pathway. SDHB-overexpression might be a new approach to inhibit tumor progression in human ovarian carcinoma. [ABSTRACT FROM AUTHOR]

Background Extensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) and derivatives are critical to the defense response against necrotrophic pathogens. Several reports demonstrate that SA limits nematode reproduction. Results Here we translate knowledge gained from studies using Arabidopsis to soybean. The ability of thirty-one Arabidopsis genes encoding important components of SA and JA synthesis and signaling in conferring resistance to soybean cyst nematode (SCN: Heterodera glycines) are investigated. We demonstrate that overexpression of three of thirty-one Arabidoposis genes in transgenic soybean roots of compost plants decreased the number of cysts formed by SCN to less than 50% of those found on control roots, namely AtNPR1(33%), AtTGA2 (38%), and AtPR-5 (38%). Three additional Arabidopsis genes decreased the number of SCN cysts by 40% or more: AtACBP3 (53% of the control value), AtACD2 (55%), and AtCM-3 (57%). Other genes having less or no effect included AtEDS5 (77%), AtNDR1 (82%), AtEDS1 (107%), and AtPR-1 (80%), as compared to control. Overexpression of AtDND1 greatly increased susceptibility as indicated by a large increase in the number of SCN cysts (175% of control). Conclusions Knowledge of the pathogen defense system gained from studies of the model system, Arabidopsis, can be directly translated to soybean through direct overexpression of Arabidopsis genes. When the genes, AtNPR1, AtGA2, and AtPR-5, encoding specific components involved in SA regulation, synthesis, and signaling, are overexpressed in soybean roots resistance to SCN is enhanced. This demonstrates functional compatibility of some Arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes. [ABSTRACT FROM AUTHOR]

Background: In the past few decades, microalgae biofuel has become one of the most interesting sources of renewable energy. However, the higher cost of microalgae biofuel compared to that of petroleum prevented microalgae biofuel production. Therefore, the research on increasing lipid productivity from microalgae becomes more important. The lipid production source, triacylglycerol biosynthesis in microalgae requires short chain fatty acids as substrates, which are synthesized in chloroplasts. However, the transport mechanism of fatty acids from microalgae chloroplasts to cytosol remains unknown. Results: cDNAs from two homologs of the Arabidopsis fatty acid exporter 1 (FAX1) were cloned from Chlamydomonas reinhardtii and were named crfax1 and crfax2. Both CrFAXs were involved in fatty acid transport, and their substrates were mainly C16 and C18 fatty acids. Overexpression of both CrFAXs increased the accumulation of the total lipid content in algae cells, and the fatty acid compositions were changed under normal TAP or nitrogen deprivation conditions. Overexpression of both CrFAXs also increased the chlorophyll content. The MGDG content was decreased but the TAG, DAG, DGDG and other lipid contents were increased in CrFAXs overexpression strains. Conclusion: These results reveal that CrFAX1 and CrFAX2 were involved in mediating fatty acid export for lipids biosynthesis in C. reinhardtii. In addition, overexpression of both CrFAXs obviously increased the intracellular lipid content, especially the triacylglycerol content in microalgae, which provides a potential technology for the production of more biofuels using microalgae. [ABSTRACT FROM AUTHOR]

Phosphorus is a poorly bioavailable macronutrient that is essential for crop growth and yield. Overuse of phosphorus fertilizers results in low phosphorus use efficiency (PUE), has serious environmental consequences and accelerates the depletion of phosphorus mineral reserves. It has become extremely challenging to improve PUE while preserving global food supplies and maintaining environmental sustainability. Molecular and genetic analyses have revealed the primary mechanisms of phosphorus uptake and utilization and their relationships to phosphorus transporters, regulators, root architecture, metabolic adaptations, quantitative trait loci, hormonal signaling and microRNA. The ability to improve PUE requires a transition from this knowledge of molecular mechanisms and plant architecture to practical strategies. These could include: i) the use of arbuscular mycorrhizal fungal symbioses for efficient phosphorus mining and uptake; ii) intercropping with suitable crop species to achieve phosphorus activation and mobilization in the soil; and iii) tissue-specific overexpression of homologous genes with advantageous agronomic properties for higher PUE along with breeding for phosphorus-efficient varieties and introgression of key quantitative trait loci. More effort is required to further dissect the mechanisms controlling phosphorus uptake and utilization within plants and provide new insight into the means to efficiently improve PUE.