Enhanced Lutein Production in Chlamydomonas reinhardtii by Overexpression of the Lycopene Epsilon Cyclase Gene.

Chlamydomonas reinhardtii is a well-established microalgal model species with a shorter doubling time, which is a promising natural source for the efficient production of high-value carotenoids. In the microalgal carotenoid biosynthetic pathway, lycopene is converted either into ß-carotene by lycopene ß-cyclase or into α-carotene by lycopene ε-cyclase (LCYE) and lycopene ß-cyclase. In this study, we overexpressed the LCYE gene in C. reinhardtii to estimate its effect on lycopene metabolism and lutein production. Chlamydomonas transformants (CrLCYE#L1, #L5, and #L6) produced significantly increased amounts of lutein per culture (up to 2.6-fold) without a decrease in cell yields. Likewise, the expression levels of LCYE gene in transformants showed a significant increase compared with that of the wild-type strain. These results suggest that LCYE overexpression enhances the conversion of lycopene to α-carotene, which in turn improves lutein productivity. Interestingly, their ß-carotene productivity appeared to increase slightly rather than decrease. Considering that the inhibition of the lycopene cyclization steps often induces higher expression in genes upstream of metabolic branches, this result implies that the redirection from ß-carotene to α-carotene by LCYE overexpression might also enhance upstream gene expression, thereby leading to auxiliary ß-carotene production.

Overexpression of the DOF-Type Transcription Factor Enhances Lipid Synthesis in Chlorella vulgaris.

In the present study, DNA binding with one finger (DOF)-type transcription factors were screened from the Chlorella vulgaris genome database. The candidate DOF transcription factor was endogenously overexpressed in C. vulgaris to improve neutral lipid production. The protein expression vector contains the heat shock protein 70 and ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit promoters and self-cleaving 2A peptide to reduce the transgene-silencing effect of C. vulgaris. A total of 74 phleomycin-resistant transformants were obtained. Under nitrogen-deficient conditions, the transformant CvDOF#3 showed approximately 1.5-fold higher neutral lipid content per cell compared to the original strain and also showed a His-tagged DOF candidate protein expression of 0.6%. Microscopic observations revealed that CvDOF#3 cells were larger in size. However, the observed differences in average cell diameter between CvDOF#3 and control cells were not statistically significant. These results indicated that the protein expression vector harboring the dual promoters and the 2A peptide, when used in combination with enzymatic cell wall degradation and glass bead transformation, could be useful for transgene and protein expression in C. vulgaris. Further experiment is necessary to confirm the expression efficiency of the HSP70 and RBCS dual promoter and 2A peptide strategy after construction of homologous recombination system in C. vulgaris. Our findings suggested that the overexpression of the endogenous DOF-type transcription factor can be used for improving the lipid content in C. vulgaris.

Synthesis, Structure, Optical, and Electrochemical Properties of Triple- and Quadruple-Decker Co-facial Tetrathiafulvalene Arrays.

Understanding the details of the electronic structure in face-to-face arranged tetrathiafulvalenes (TTFs) is very important for the design of supramolecular functional materials and superior conductive organic materials. This article is a comprehensive study of the interactions among columnar stacked TTFs using trimeric (trimer) and tetrameric (tetramer) TTFs linked by alkylenedithio groups (-S(CH2 )n S-, n=1-4) as models of triple- and quadruple-decker TTF arrays. Single-crystal X-ray analyses of neutral trimeric TTFs revealed that the three TTF moieties are oriented in a zigzag arrangement. Cyclic voltammetry measurements (CV) reveal that the trimer and tetramer exhibited diverse reversible redox processes with multi-electron transfers, depending on the length of the -S(CH2 )n S- units and substituents. The electronic spectra of the radical cations, prepared by electrochemical oxidation, showed charge resonance (CR) bands in the NIR/IR region (1630-1850 nm), attributed to a mixed valence (MV) state of the triple- and quadruple-decker TTF arrays. In the trimeric systems, the dicationic state (+2; 0.66 cation per TTF unit) was found to be a stable state, whereas the monocationic state (+1) was not observed in the electronic spectra. In the tetrameric system, substituent-dependent redox processes were observed. Moreover, π-trimers and π-tetramers, which show a significant Davydov blueshift in the spectra, are formed in the tricationic (trimer) and tetracationic (tetramer) state. In addition, these attractive interactions are strongly dependent on the length of the linkage unit.

The phenylic hydroxyl group is essential for the induction of stress response by sodium salicylate.

We have shown that sodium salicylate (SA) activates the heat shock promoter and induces the expression of heat shock proteins (Hsps) with a concomitant increase in the thermotolerance of cells. To identify the functional groups of SA necessary for the induction of Hsps, we evaluated the effect of various derivatives of SA using a mammalian cell line containing a reporter gene downstream of an hsp105 promoter. Among the derivatives, the compounds in which the carboxyl group of SA was substituted activated the hsp105 promoter at 37 degrees C as SA did, but the compounds in which the hydroxyl group was substituted did not. Thus, the phenylic hydroxyl group but not the carboxyl group of SA seemed to be necessary for a stress-induced response. In addition, the orientation of two functional groups on the benzene ring of SA derivatives was also important for the induction of a response. Among these compounds, salicylalcohol which strongly induced the expression of Hsps suppressed the protein aggregation and apoptosis caused by an expanded polyglutamine tract in a cellular model of polyglutamine disease. These findings may aid in the development of novel effective Hsp-inducers.