Expression of C-5 sterol desaturase from an edible mushroom in fisson yeast enhances its ethanol and thermotolerance.

Bioethanol is an environment friendly and renewable source of energy produced by the fermentation of agricultural raw material by a variety of microorganisms including yeast. Obtaining yeast strains that are tolerant to stresses like high levels of ethanol and high temperature is highly desirable as it reduces cost and increases yield during bioethanol production. Here, we report that heterologous expression of C-5 Sterol desaturase (FvC5SD)-an ergosterol biosynthesis enzyme from an edible mushroom Flammulina velutipes in fission yeast, not only imparts increased thermotolerance but also tolerance towards high ethanol concentration and low pH. This tolerance could be attributed to an increase of ≈1.5 fold in the level of ergosterol and oleic acid (C-18 unsaturated fatty acid) as analysed by gas chromatography- mass spectrometry. FvC5SD is a membrane localized iron binding enzyme that introduces double bond at C-5 position into the Δ7-sterol substrates to yield Δ5, 7- sterols as products. In F. velutipes, FvC5SD transcript was observed to be upregulated by ≈5 fold under low pH condition and by ≈ 9 folds and ≈5 fold at 40°C and 4°C respectively when compared to normal growth temperature of 23°C. Besides, susceptibility to cell wall inhibiting drugs like Congo red and Calcoflour white was also found to increase in FvC5SD expressing S. pombe strain. Alteration in membrane sterol and fatty acid composition could also lead to increase in susceptibility to cell wall inhibiting drugs. Thus, this study has immense industrial application and can be employed to ensure competitiveness of fermentation process.

Genetically modified (GM) crops: milestones and new advances in crop improvement.

KEY MESSAGE: New advances in crop genetic engineering can significantly pace up the development of genetically improved varieties with enhanced yield, nutrition and tolerance to biotic and abiotic stresses. Genetically modified (GM) crops can act as powerful complement to the crops produced by laborious and time consuming conventional breeding methods to meet the worldwide demand for quality foods. GM crops can help fight malnutrition due to enhanced yield, nutritional quality and increased resistance to various biotic and abiotic stresses. However, several biosafety issues and public concerns are associated with cultivation of GM crops developed by transgenesis, i.e., introduction of genes from distantly related organism. To meet these concerns, researchers have developed alternative concepts of cisgenesis and intragenesis which involve transformation of plants with genetic material derived from the species itself or from closely related species capable of sexual hybridization, respectively. Recombinase technology aimed at site-specific integration of transgene can help to overcome limitations of traditional genetic engineering methods based on random integration of multiple copy of transgene into plant genome leading to gene silencing and unpredictable expression pattern. Besides, recently developed technology of genome editing using engineered nucleases, permit the modification or mutation of genes of interest without involving foreign DNA, and as a result, plants developed with this technology might be considered as non-transgenic genetically altered plants. This would open the doors for the development and commercialization of transgenic plants with superior phenotypes even in countries where GM crops are poorly accepted. This review is an attempt to summarize various past achievements of GM technology in crop improvement, recent progress and new advances in the field to develop improved varieties aimed for better consumer acceptance.

A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter.

Oxalate decarboxylase (OXDC) enzyme has immense biotechnological applications due to its ability to decompose anti-nutrient oxalic acid. Flammulina velutipes, an edible wood rotting fungus responds to oxalic acid by induction of OXDC to maintain steady levels of pH and oxalate anions outside the fungal hyphae. Here, we report that upon oxalic acid induction, a calmodulin (CaM) like protein-FvCaMLP, interacts with the OXDC promoter to regulate its expression. Electrophoretic mobility shift assay showed that FvCamlp specifically binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter. Moreover, substitutions of amino acids in the EF hand motifs resulted in loss of DNA binding ability of FvCamlp. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript pointing towards positive nature of the regulation. FvCaMLP is different from other known EF hand proteins. It shows sequence similarity to both CaMs and myosin regulatory light chain (Cdc4), but has properties typical of a calmodulin, like binding of (45)Ca(2+), heat stability and Ca(2+) dependent electrophoretic shift. Hence, FvCaMLP can be considered a new addition to the category of unconventional Ca(2+) binding transcriptional regulators.

Small RNAs in plants: recent development and application for crop improvement.

The phenomenon of RNA interference (RNAi) which involves sequence-specific gene regulation by small non-coding RNAs, i.e., small interfering RNA (siRNA) and microRNA (miRNA) has emerged as one of most powerful approaches for crop improvement. RNAi based on siRNA is one of the widely used tools of reverse genetics which aid in revealing gene functions in many species. This technology has been extensively applied to alter the gene expression in plants with an aim to achieve desirable traits. RNAi has been used for enhancing the crop yield and productivity by manipulating the gene involved in biomass, grain yield and enhanced shelf life of fruits and vegetables. It has also been applied for developing resistance against various biotic (bacteria, fungi, viruses, nematodes, insects) and abiotic stresses (drought, salinity, cold, etc.). Nutritional improvements of crops have also been achieved by enriching the crops with essential amino acids, fatty acids, antioxidants and other nutrients beneficial for human health or by reducing allergens or anti-nutrients. microRNAs are key regulators of important plant processes like growth, development, and response to various stresses. In spite of similarity in size (20-24 nt), miRNA differ from siRNA in precursor structures, pathway of biogenesis, and modes of action. This review also highlights the miRNA based genetic modification technology where various miRNAs/artificial miRNAs and their targets can be utilized for improving several desirable plant traits. microRNA based strategies are much efficient than siRNA-based RNAi strategies due to its specificity and less undesirable off target effects. As per the FDA guidelines, small RNA (sRNA) based transgenics are much safer for consumption than those over-expressing proteins. This review thereby summarizes the emerging advances and achievement in the field of sRNAs and its application for crop improvement.

Characterization of a putative spindle assembly checkpoint kinase Mps1, suggests its involvement in cell division, morphogenesis and oxidative stress tolerance in Candida albicans.

In Saccharomyces cerevisiae MPS1 is one of the major protein kinase that governs the spindle checkpoint pathway. The S. cerevisiae structural homolog of opportunistic pathogen Candida albicans CaMPS1, is indispensable for the cell viability. The essentiality of Mps1 was confirmed by Homozygote Trisome test. To determine its biological function in this pathogen conditional mutant was generated through regulatable MET3 promoter. Examination of heterozygous and conditional (+Met/Cys) mps1 mutants revealed a mitosis specific arrest phenotype, where mutants showed large buds with undivided nuclei. Flowcytometry analysis revealed abnormal ploidy levels in mps1 mutant. In presence of anti-microtubule drug Nocodazole, mps1 mutant showed a dramatic loss of viability suggesting a role of Mps1 in Spindle Assembly Checkpoint (SAC) activation. These mutants were also defective in microtubule organization. Moreover, heterozygous mutant showed defective in-vitro yeast to hyphae morphological transition. Growth defect in heterozygous mutant suggest haploinsufficiency of this gene. qRT PCR analysis showed around 3 fold upregulation of MPS1 in presence of serum. This expression of MPS1 is dependent on Efg1 and is independent of other hyphal regulators like Ras1 and Tpk2. Furthermore, mps1 mutants were also sensitive to oxidative stress. Heterozygous mps1 mutant did not undergo morphological transition and showed 5-Fold reduction in colony forming units in response to macrophage. Thus, the vital checkpoint kinase, Mps1 besides cell division also has a role in morphogenesis and oxidative stress tolerance, in this pathogenic fungus.

Upregulation of galactose metabolic pathway by N-acetylglucosamine induced endogenous synthesis of galactose in Candida albicans.

N-Acetylglucosamine (GlcNAc) is an important signaling molecule that plays multiple roles in Candida albicans. Induction of galactose metabolic pathway by GlcNAc is an intriguing aspect of C. albicans biology. In order to investigate the role of galactose metabolic genes (GAL genes) in presence of GlcNAc, we created knockouts of galactokinase (GAL1) and UDP galactose epimerase (GAL10) genes. These mutants failed to grow on galactose and also showed lower growth rate in presence of GlcNAc. Interestingly, expression of GAL genes in presence of GlcNAc was higher in gal1Δ strain relative to that of wild type strain. Moreover, no GlcNAc induced upregulation of GAL genes was observed in the gal10Δ strain suggesting that UDP galactose epimerase is essential for GlcNAc induced activation of GAL genes. GlcNAc induced expression of GAL genes was also investigated in GlcNAc metabolic pathway triple mutant N216 (hxk1Δ nag1Δ dac1Δ). Interestingly, in this mutant the GAL genes are neither induced nor repressed and remain derepressed as found on a neutral carbon source such as glycerol, suggesting that catabolism of GlcNAc play an important role in the expression of GAL genes. GC/MS analysis of derivatized metabolites revealed a significant accumulation of galactose in the gal1Δ strain while no galactose was detected in gal10Δ and N216 strain. Solution-state NMR spectroscopy using N-acetyl-¹³C1-glucosamine confirmed the flow of ¹³C label from GlcNAc to galactose. Thus, internal galactose synthesized via UDP galactose pathway from GlcNAc metabolites acts as the inducer of GAL genes in presence of GlcNAc.

Expression of a fungal sterol desaturase improves tomato drought tolerance, pathogen resistance and nutritional quality.

Crop genetic engineering mostly aims at improving environmental stress (biotic and abiotic) tolerance as well as nutritional quality. Empowering a single crop with multiple traits is highly demanding and requires manipulation of more than one gene. However, we report improved drought tolerance and fungal resistance along with the increased iron and polyunsaturated fatty acid content in tomato by expressing a single gene encoding C-5 sterol desaturase (FvC5SD) from an edible fungus Flammulina velutipes. FvC5SD is an iron binding protein involved in ergosterol biosynthesis. Morphological and biochemical analyses indicated ≈23% more epicuticular wax deposition in leaves of transgenic plants that provides an effective waterproof barrier resulting in improved protection from drought and infection by phytopathogenic fungus Sclerotiniasclerotiorum. Furthermore, the transgenic fruits have improved nutritional value attributed to enhanced level of beneficial PUFA and 2-3 fold increase in total iron content. This strategy can be extended to other economically important crops.