Delineating the molecular responses of a halotolerant microalga using integrated omics approach to identify genetic engineering targets for enhanced TAG production

Abstract Background Harnessing the halotolerant characteristics of microalgae provides a viable alternative for sustainable biomass and triacylglyceride (TAG) production. Scenedesmus sp. IITRIND2 is a fast growing fresh water microalga that has the capability to thrive in high saline environments. To understand the microalga’s adaptability, we studied its physiological and metabolic flexibility by studying differential protein, metabolite and lipid expression profiles using metabolomics, proteomics, real-time polymerase chain reaction, and lipidomics under high salinity conditions. Results On exposure to salinity, the microalga rewired its cellular reserves and ultrastructure, restricted the ions channels, and modulated its surface potential along with secretion of extrapolysaccharide to maintain homeostasis and resolve the cellular damage. The algal-omics studies suggested a well-organized salinity-driven metabolic adjustment by the microalga starting from increasing the negatively charged lipids, up regulation of proline and sugars accumulation, followed by direction of carbon and energy flux towards TAG synthesis. Furthermore, the omics studies indicated both de-novo and lipid cycling pathways at work for increasing the overall TAG accumulation inside the microalgal cells. Conclusion The salt response observed here is unique and is different from the well-known halotolerant microalga; Dunaliella salina, implying diversity in algal response with species. Based on the integrated algal-omics studies, four potential genetic targets belonging to two different metabolic pathways (salt tolerance and lipid production) were identified, which can be further tested in non-halotolerant algal strains

Insights into the Enhanced Lipid Production Characteristics of a Fresh Water Microalga under High Salinity Conditions

Bioprospecting of microalgae capable of growing and accumulating high amounts of lipids in high salinity conditions such as seawater can substantially improve the economic vaibaility of algal biodiesel production. In view of this, a fresh water microalga, <i>Scenedesmus</i> sp. IITRIND2, was cultivated under saline conditions to assess its halotolerant behavior and potential as biodiesel feedstock. The microalga efficiently adapted to 100% seawater salinity, enhanced its lipid content by 52%, thus yielded ∼3.2 fold higher lipid productivity as compared to the Bold’s basal media (BBM). The increase in the lipid content was balanced by a sharp decrease in its protein and carbohydrate content. Biochemical analysis evidenced that salinity induced oxidative stress resulted in reduced levels of photosynthetic pigments, elevated levels of reactive oxygen species (H₂O₂, thiobarbituric acid reactive substances), osmolytes (proline, glycine betaine), and activity of antioxidant enzymes (catalase, ascorbate peroxidase). These studies suggested that microalga efficiently modulated its metabolic flexibility in order to acclatamize the salanity induced stress. Further, the FAME analysis revealed the dominance of C14:0, C16:0, C18:0, C18:1, and C18:2 fatty acids under halotolerant conditions, and the properties of the resulting biodiesel were in compliance with ASTM (American Society for Testing Materials) D6751 and EN 14214 (European) fuel standards. These results consolidate that the lipid augmented halotolerant algal strains capable of growing in saline/seawater can be formulated as environmental sustainable and economic viable sources for biodiesel production

Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation

<p>Abstract</p> <p>Background</p> <p>Viral vectors provide a method of stably introducing exogenous DNA into cells that are not easily transfectable allowing for the ectopic expression or silencing of genes for therapeutic or experimental purposes. However, some cell types, in particular bone marrow cells, dendritic cells and neurons are difficult to transduce with viral vectors. Successful transduction of such cells requires preparation of highly concentrated viral stocks, which permit a high virus concentration and multiplicity of infection (MOI) during transduction. Pseudotyping with the vesicular stomatitis virus G (VSV-G) envelope protein is common practice for both lentiviral and retroviral vectors. The VSV-G glycoprotein adds physical stability to retroviral particles, allowing concentration of virus by high-speed ultracentrifugation. Here we describe a method report for concentration of virus from large volumes of culture supernatant by means of successive rounds of ultracentrifugation into the same ultracentrifuge tube.</p> <p>Method</p> <p>Stable retrovirus producer cell lines were generated and large volumes of virus-containing supernatant were produced. We then tested the transduction ability of virus following varying rounds of concentration by ultra-centrifugation. In a second series of experiments lentivirus-containing supernatant was produced by transient transfection of 297T/17 cells and again we tested the transduction ability of virus following multiple rounds of ultra-centrifugation.</p> <p>Results</p> <p>We report being able to centrifuge VSV-G coated retrovirus for as many as four rounds of ultracentrifugation while observing an additive increase in viral titer. Even after four rounds of ultracentrifugation we did not reach a plateau in viral titer relative to viral supernatant concentrated to indicate that we had reached the maximum tolerated centrifugation time, implying that it may be possible to centrifuge VSV-G coated retrovirus even further should it be necessary to achieve yet higher titers for specific applications. We further report that VSV-G coated lentiviral particles may also be concentrated by successive rounds of ultracentrifugation (in this case four rounds) with minimal loss of transduction efficiency.</p> <p>Conclusion</p> <p>This method of concentrating virus has allowed us to generate virus of sufficient titers to transduce bone marrow cells with both retrovirus and lentivirus, including virus carrying shRNA constructs.</p