A lycopene β‐cyclase/lycopene ε‐cyclase/light‐harvesting complex‐fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α‐carotene and β‐carotene at different ratios

Biosynthesis of asymmetric carotenoids such as α‐carotene and lutein in plants and green algae involves the two enzymes lycopene β‐cyclase (LCYB) and lycopene ε‐cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C‐terminal light‐harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α‐carotene and β‐carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C‐terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α‐carotene‐to‐β‐carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α‐carotene or lutein in bacteria or fungi.     

THE RELATIONSHIP BETWEEN PSEUDOSCOURFIELDIA MARINA AND PYCNOCOCCUS PROVASOLII (PRASINOPHYCEAE, CHLOROPHYTA): EVIDENCE FROM 18S rDNA SEQUENCE DATA

18S rDNA sequences were obtained for the prasinophytes Pseudoscourfieldia marina (Throndsen) Manton and Pycnococcus provasolii Guillard. These sequences, along with those of additional prasinophytes and other green algae and land plants, were used for phylogenetic analyses by the neighbor-joining, maximum parsimony, and quartet puzzling methods. Results indicate that Ps. marina and Py. provasolii are closely allied and that both should be included in the Pycnococcaceae. In addition, results of these sequence analyses and additional pigment analysis indicate that the organism previously identified as Ps. marina, isolate CCMP 717, is, in fact, a Nephroselmis sp. or is closely related to that genus.     

PHOTOSYNTHETIC PIGMENTS OF PSEUDOSCOURFIELDIA MARINA AND SELECT GREEN FLAGELLATES AND COCCOID ULTRAPHYTOPLANKTON: IMPLICATIONS FOR THE SYSTEMATICS OF THE MICROMONADOPHYCEAE (CHLOROPHYTA)1

Photosynthetic pigments of the green flagellate Pseudoscourfieldia marina (Throndsen) Manton (Micromonadophyceae) are similar to those of the coccoid Pycnococcus provasolii Guillard; prasinoxanthin is the predominant carotenoid. Other organisms that possess prasinoxanthin also possess additional pigments not found in either P. marina or P. provasolii. Uriolide, a xanthophyll previously described from the coccoid done URI 266G, was also found in Mantoniella squamata (Manton et Parke) Desikachary, Micromonas pusilla Manton et Parke and Mamiella gilva (Parks et Rayns) Moestrup, all flagellate members of the Mamiellales, and the coccoid clone IV E5G. Other unidentified carotenoids were also present in M. squamata, M. pusilla, and M. gilva. These results suggest that P. marina and the coccoid organisms URI 266G and IV E5G may be related to the Mamiellales, and that P. provasolii may be more closely related to P. marina than to M. squamata, M. pusilla, and M. gilva.     

PHYLOGENETIC POSITION OF CRUSTOMASTIX STIGMATICA SP. NOV. AND DOLICHOMASTIX TENUILEPIS IN RELATION TO THE MAMIELLALES (PRASINOPHYCEAE,CHLOROPHYTA)1

A new marine microalga from the Mediterranean Sea, Crustomastix stigmatica Zingone, is investigated by means of LM, SEM, TEM, and pigment and molecular analyses (nuclear‐encoded small subunit [SSU] rDNA and plastid‐encoded rbcL). Pigment and molecular information is also provided for the related species Dolichomastix tenuilepis Throndsen et Zingone. Crustomastix stigmatica has a bean‐shaped cell body 3–5 μm long and 1.5–2.8 μm wide, with two flagella four to five times the body length. The single chloroplast is pale yellow‐green, cup‐shaped, and lacks a pyrenoid. A small bright yellow stigma is located in the mid‐dorsal part of the cell under the chloroplast membrane. An additional accumulation of osmiophilic globules is at times seen in a chloroplast lobe. Cells lack flat scales, whereas three different types of hair‐like scales are present on the flagella. The main pigments of C. stigmatica are those typical of Mamiellales, though siphonein/siphonaxanthin replaces prasinoxanthin and uriolide is absent. The pigment pool of D. tenuilepis is more similar to that of Micromonas pusilla (Butcher) Manton et Parke and of other Mamiellales. The nuclear SSU rDNA phylogeny shows that the inclusion of C. stigmatica and D. tenuilepis in the Mamiellales retains monophyly for the order. The two species form a distinct clade, which is sister to a clade including all the other Mamiellales. Results of rbcL analyses failed to provide phylogenetic information at both the order and species level. No unique morphological or pigment characteristics circumscribe the mamiellalean clade as a whole nor its two daughter clades.     

Recovery of 197 eukaryotic bins reveals major challenges for eukaryote genome reconstruction from terrestrial metagenomes

As most eukaryotic genomes are yet to be sequenced, the mechanisms underlying their contribution to different ecosystem processes remain untapped. Although approaches to recovering Prokaryotic genomes have become common in genome biology, few studies have tackled the recovery of eukaryotic genomes from metagenomes. This study assessed the reconstruction of microbial eukaryotic genomes using 6000 metagenomes from terrestrial and some transition environments using the EukRep pipeline. Only 215 metagenomic libraries yielded eukaryotic bins. From a total of 447 eukaryotic bins recovered 197 were classified at the phylum level. Streptophytes and fungi were the most represented clades with 83 and 73 bins, respectively. More than 78% of the obtained eukaryotic bins were recovered from samples whose biomes were classified as host-associated, aquatic, and anthropogenic terrestrial. However, only 93 bins were taxonomically assigned at the genus level and 17 bins at the species level. Completeness and contamination estimates were obtained for a total of 193 bins and consisted of 44.64% (σ = 27.41%) and 3.97% (σ = 6.53%), respectively. Micromonas commoda was the most frequent taxon found while Saccharomyces cerevisiae presented the highest completeness, probably because more reference genomes are available. Current measures of completeness are based on the presence of single-copy genes. However, mapping of the contigs from the recovered eukaryotic bins to the chromosomes of the reference genomes showed many gaps, suggesting that completeness measures should also include chromosome coverage. Recovering eukaryotic genomes will benefit significantly from long-read sequencing, development of tools for dealing with repeat-rich genomes, and improved reference genomes databases.     

PIGMENTS OF BATHYCOCCUS PRASINOS (PRASINOPHYCEAE): METHODOLOGICAL AND CHEMOSYSTEMATIC IMPLICATIONS1,2

Bathycoccus prasinos Eikrem et Throndsen exhibited a complex carotenoid distribution pattern including the carotenes β,β-carotene (0.8% of total carotenoids) and β, ° Carotene (0.4%) and several xanthophylls. These were prasinoxanthin (49% of total carotenoids), micromonal (16%), neoxanthin (14%), uriolide (7%), violaxanthin (0.8%), 3¹-dehydrouriolide (0.8%), dihydrolutein (0.1%), two partly characterized esterified carotenols (together 10%), and five minor unidentified carotenols (together 2%). The identifications were based on high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), visible spectroscopy (VIS), and mass spectra (MS) and in part on ¹H nuclear magnetic resonance (NMR), circular dichroism (CD), and chemical derivatization. The carotenoid composition of B. prasinos was related to that of other prasinoxanthin / uriolide / micromonal-producing prasinophytes (Mantoniella squamata, Micromonas pusilla, and Pseudoscourfieldia marina). The relative distribution of chlorophylls (w/w) were chlorophyll a (chl a; 63%), chl b (31%), and an unknown chl c-like chlorophyll (7%) with spectral characteristics similar to magnesium 2,4-divinylphaeoporphyrin a, monomethyl ester, compatible with other prasinophytes. The chemosystematic data and ultrastructural characteristics for the order Mamiellales are discussed. We conclude that HPLC studies alone are insufficient for the identification and characterization of the carotenoids, including the minor carotenoids essential for biosynthetic/chemosystematic considerations.