Impact of Different Light Characteristics on the Growth and Lipid Content of Diatom Phaeodactylum tricornutum Transconjugant Strains

Light regulates important metabolic processes in microalgal cells, which can further impact the metabolism and the accumulation of biomolecules such as lipids, carbohydrates, and proteins. Different characteristics of light have been studied on various strains of the model diatom Phaeodactylum tricornutum, but not on transconjugant cells and information on wild-type strains is still limited. Therefore, we studied the impact of different light characteristics such as spectral quality, light intensity and light shift on the growth, and the composition in lipids and fatty acids of P. tricornutum cells to provide a comprehensive context for future applications. Initially, we tested the impact of spectral quality and light intensity on P. tricornutum transformed with an episomal vector (Ptev), harboring the resistance gene Sh ble. Results indicated that Ptev cells accumulated more biomass and overall lipids in spectral quality Red 1 (R1: 34% > 600 nm > 66%) more effectively as compared to Red 2 (R2: 8% > 600 nm > 92%). It was also detected that cell granularity was higher in R1 as compared to R2. Furthermore, by testing two light intensities 65 μmol·m-2·s-1 and 145 μmol·m-2·s-1 light, it was observed that 145 μmol·m-2·s-1 led to an increase in growth trend, total biomass and lipid content. Combining spectral qualities and light intensities, we show that the lipid accumulation raised by 2.8-fold. Studying the light intensity and spectral quality allowed us to optimize the light conditions to R1 spectral quality and light intensity 145 μmol·m-2·s-1. These initial results showed that red light R1 at 145 μmol·m-2·s-1 was the best condition for biomass and total lipids accumulation in Ptev cells. Next, we further combined these two-light optimizations with a third light characteristics, i.e. light shift, where the cultures were shifted during the early stationary phase to a different light environment. We studied Red light shift (Rs) to investigate how light condition variations impacted P. tricornutum transconjugants Ptev and with an episomal vector containing the reporter gene YFP (PtYFP). We observed that Rs induced growth and fatty acid eicosapentaenoic acid (EPA) in Ptev as compared to PtYFP. Altogether, the study shows that red light shift of R1 at 145 μmol·m-2·s-1 promoted biomass and total lipids accumulation in Ptev and PtYFP cells. The study provides a comprehensive approach to using different light characteristics with the aim to optimize growth and lipids, as well as to fatty acid production.

Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells

Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral(NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region(S/MAR) sequence(SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore,Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion,the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.