Efficient heterologous transformation of Chlamydomonas reinhardtii npq2 mutant with the zeaxanthin epoxidase gene isolated and characterized from Chlorella zofingiensis.

In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light.

The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene ß-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and ß-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

Enhancement of lutein production in Chlorella sorokiniana (Chorophyta) by improvement of culture conditions and random mutagenesis.

Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 µmol photons m(-2) s(-1), 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N'-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L(-1) and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g(-1) dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.

Tres nuevos registros del genero Carex (Cyperaceae) para el Perú y adiciones a la flora andina del departamento de Huánuco / Three new records of the genus Carex (Cyperaceae) from Peru and additions to the Andean flora of the Huánuco department

Three new records from Peru (Carex humahuacaensis G.A. Wheeler, Carex peucophila T. Holm y Carex pygmaea Boeck.), and 75 new records of vascular plants are reported for Huánuco department. Its habitat, distribution and the conservation status of endemisms such as: Caiophora cirsiifolia C. Presl, Carex hebetata Boott and Senecio macrorrhizus Wedd. are commented.

Tres nuevos registros para el Perú (Carex humahuacaensis G.A. Wheeler, Carex peucophila T. Holm y Carex pygmaea Boeck.) y 75 nuevos registros para la flora del departamento de Huánuco son dados a conocer. Se informan datos sobre el hábitat, distribución y en el caso de endemismos como: Caiophora cirsiifolia C. Presl, Carex hebetata Boott, y Senecio macrorrhizus Wedd. su estado actual de conservación.

Surface modification of H-ferrierite by reaction with triethoxysilane.

The interaction of triethoxysilane (TES) with H-ferrierite (H-FER) and its effects on acidity have been investigated by infrared spectroscopy. TES adsorbs only on the external surface of H-FER and allows the almost complete disappearance of the external silanol groups. New SiH groups are formed which appear to be inactive in acid-base interactions. The adsorption of propionitrile, which diffuses into the zeolitic channels, provides evidence for the lack of substantial perturbation of the strongly acidic internal bridging OH groups. On the contrary, the adsorption of the hindered basic probe molecule o-toluonitrile, which cannot penetrate the FER channels, shows that not only terminal silanols but also Al3+ Lewis acid sites present on the external surface of H-FER almost totally disappear after TES treatment. Treatment with TES seems to allow virtually the total deactivation of the H-FER external surface.