The potential of production of sulfated polysaccharides from Porphyridium

Summary The environmental conditions prevailing in Israel make marine algae an attractive crop for the production of valuable chemicals. A marine species of Porphyridium seems to fit this purpose.The unicellular red alga Porphyridium is encapsulated by a polysaccharide envelope that is present in the gel state. This polysaccharide is an acidic heteropolymer composed of sulfated sugars. It forms ionic bridges through divalent cations, thus reaching a very high molecular weight. The thickness of the polysaccharide capsule varies according to the phase of growth and the growth conditions. Its outer part dissolves in the growth medium, which becomes progressively more viscous. Sulfated polysaccharides form theramlly reversible gels similar to agar and carrageenan, which are usually extracted from marine macroalgae. These gels have been finding increasing use in commercial applications as gelling agents, thickeners, stabilizers, and emulsifiers.We have done experiments on the cultivation of a marine species of Porphyridium for the production of polysaccharides. This unicellular alga has an advantage over the macroalgae due to its relatively faster growth rate and the possibility to regulate its growth. The potential for production of the polysaccharide, both that dissolved in the external medium and that attached to the cell (including an intracellular fraction), and the effects of growth conditions on productivity were suudied in the laboratory. Porphyridium was also cultivated outdoors in seawater in 1-m² ponds and its growth potential investigated.     

The amino acid sequences of the cytochromes c553 from Porphyridium cruentum and Aphanizomenon flos-aquae

The amino acid sequences of cytochrome c₅₅₃ from the eukaryotic red alga Porphyridium cruentum and from the prokaryotic cyanobacterium Aphanizomenon flos-aquae have been determined from the tryptic and cyanogen bromide peptides. The results indicate that a charged region of these proteins has evolved with special rapidity to accomodate a rapid evolution of a binding site in the P₇₀₀ electron acceptor complex.     

The Supramolecular Structure of Photosystem II — Phycobilisome-Complexes of Porphyridium cruentum

The structure and arrangement of phycobilisomes of the unicellular red alga Porphyridium cruentum is compared with the organization of the thylakoid freeze-fracture particles in order to determine the relationship between phycobilisomes and photosystem II. The hemi-ellipsoidal phycobilisomes, 20 nm thick, are predominantly organized into rows; their centre to centre periodicity is 30–40 nm, so that they are well separated by a gap of 10–20 nm. The phycobilisomes are cleaved by a central faint furrow, parallel to the long axis from top to base. The organization of the exoplasmic particles in rows is similar to the arrangement of the phycobilisomes so that a structural relationship between both systems, previously demonstrated in cyanobacteria, is evident. Within the rows, the 10 nm EF-particles are grouped in tetrameric complexes separated by distances similar to those observed for phycobilisomes. We propose that the tetrameric EF-particle complexes correspond to tetrameric photosystem II complexes which bind one hemi-ellipsoidal phycobilisome on the stroma exposed surface of the thylakoid. A hypothetical model of this photosystem II-phycobilisome complex is presented.     

Cell fragility — The key problem of microalgae mass production in closed photobioreactors

The gap between the theoretical biological potential of microalgae and the biomass productivity obtained with algal culture in tubular biophotoreactors is due to a reduced growth rate related to hydrodynamic stress of pumping. High levels of mixing are necessary to reach a turbulent flow of the culture, in order to optimize the light regime. The optimal conditions of pumping to produce this significant liquid mixing may produce some cell damage. Factors affecting this hydrodynamic stress (geometry of the bioreactor involved, type of pump utilized, morphology of algal cells, physiological conditions of microalgae, etc.) are discussed.     

Characterization and potential antitumor effect of a heteropolysaccharide produced by the red alga Porphyridium sordidum

Taking into account the rising trend of the incidence of cancers of various organs, effective therapies are urgently needed to control human malignancies. However, almost all chemotherapy drugs currently on the market cause serious side effects. Fortunately, several studies have shown that some non‐toxic biological macromolecules, including algal polysaccharides, possess anti‐cancer activities or can increase the efficacy of conventional chemotherapy drugs. Polysaccharides are characteristic secondary metabolites of many algae. The efficacy of polysaccharides on the normal and cancer cells is not well investigated, but our investigations proved a cell specific effect of a newly isolated extracellular polysaccharide from the red microalga Porphyridium sordidum. The investigated substance was composed of xylose:glucose and galactose:manose:rhamnose in a molar ratio of 1:0.52:0.44:0.31. Reversible electroporation has been exploited to increase the transport through the plasma membrane into the tested breast cancer tumor cells MCF‐7 and MDA‐MB231. Application of 75 µg/mL polysaccharide in combination with 200 V/cm electroporation induced 40% decrease in viability of MDA‐MB231 cells and changes in cell morphology while control cells (MCF10A) remained with normal morphology and kept vitality.     

Characterization of the Porphyridium cruentum Chl a-binding LHC by in vitro reconstitution: LHCaR1 binds 8 Chl a molecules and proportionately more carotenoids than CAB proteins

The Porphyridium cruentum light harvesting complex (LHC) binds Chl a, zeaxanthin and -carotene and comprises at least 6 polypeptides of a multigene family. We describe the first in vitro reconstitution of a red algal light-harvesting protein (LHCaR1) with Chl a/carotenoid extracts from P. cruentum. The reconstituted pigment complex (rLHCaR1) is spectrally similar to the native LHC I, with an absorption maximum at 670 nm, a 77 K fluorescence emission peak at 677 nm (ex. 440 nm), and similar circular dichroism spectra. Molar ratios of 4.0 zeaxanthin, 0.3 -carotene and 8.2 Chl a per polypeptide for rLHCaR1 are similar to those of the native LHC I complex (3.1 zeaxanthin, 0.5 -carotene, 8.5 Chl a). The binding of 8 Chl a molecules per apoprotein is consistent with 8 putative Chl-binding sites in the predicted transmembrane helices of LHCaR1. Two of the putative Chl a binding sites (helix 2) in LHCaR1 were assigned to Chl b in Chl a/b-binding (CAB) LHC II [Kühlbrandt et al. (1994) Nature 367: 614–21]. This suggests either that discrimination for binding of Chl a or Chl b is not very specific at these sites or that specificity of binding sites evolved separately in CAB proteins. LHCaR1 can be reconstituted with varying ratios of carotenoids, consistent with our previous observation that the carotenoid to Chl ratio is substantially higher in P. cruentum grown under high irradiance. Also notable is that zeaxanthin does not act as an accessory light-harvesting pigment, even though it is highly likely that it occupies the position assigned to lutein in the CAB LHCs.This revised version was published online in October 2005 with corrections to the Cover Date.     

INTRASPECIFIC TRANSFER OF HERBICIDE RESISTANCE IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYCEAE) VIA PROTOPLAST FUSION

Stable progeny doubly resistant to the herbicides sulfometuron methyl (SMM) and diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] (DCMU) were obtained at a frequency of 2% on fusion of protoplasts derived from mutants of Porphyridium sp. (UTEX 637) that were resistant only to SMM (strain SMR) or DCMU (strain DC-2). In the presence of both herbicides, only the fusion progeny could grow; both parental mutants were inhibited. In the absence of SMM, the activity of acetohydroxy acid synthase (AHAS) in the wild-type strain was similar to that in DC-2, exceeding that of SMR by up to 4.5-fold. AHAS activities of all fusion progeny were lower than those of the wild-type strain and DC-2 but higher than that of SMR. In the presence of SMM, AHAS activities of all tested fusion progeny ranged between those of the two parental mutants. This result indicates that both types of AHAS, the type resistant to SMM and the sensitive type, originating from SMR and DC-2, respectively, were expressed in the fusion progeny. In the presence of DCMU, the photosynthetic activity of SMR was completely inhibited, whereas that of DC-2 was unaffected. The photosynthetic activity of the fusion progeny in the presence of DCMU was slightly lower than that of DC-2. Both the cell volume and the DNA content of the fusion progeny were similar to those of the parents. However, the genetic nature of the fusion products has not yet been elucidated. To the best of our knowledge, this is the first report on transfer of herbicide resistance via protoplast fusion in algae.     

Comparison of artificial light photobioreactors and other production systems using Porphyridium cruentum

A new type of preparative photobioreactor for high quality production of microalgae is developed for hatchery-nursery of marine animals, as well as for fine chemicals extraction. Of modular conception, two artificial light photobioreactors in plastic and stainless steel are designed so as to provide strictly controlled conditions in an attempt to increase quality and diminish cost prices. They are assessed for production of Porphyridum cruentum and compared to conventional transparent tanks and solar photobioreactors. The concentration and productivity obtained are ten-fold higher than with hatchery tanks, which leads to a significant drop in cost price of biomass. Comparison is also made with a 10 m² solar photobioreactor operated in the south of France, for which biomass cost price is half that of 1.5 m² artificial light photobioreactor. Extrapolations erasing size discrepancy show that the cost price of the two technologies are not very different. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification and quantification of indole-3-acetic acid in the kelp Laminaria japonica Areschoug and its effect on growth of marine microalgae

A method was established for the identification and quantification of indole-3-acetic acid (IAA) in extracts of the kelp Laminaria japonica. An IAA content of 90–95 μg kg⁻¹ fresh weight in kelp extract was determined by high performance liquid chromatography (HPLC). IAA identification was based on a combination of co-chromatography and comparative chromatography with a standard, analysis of UV spectra, and atmospheric pressure electrospray mass spectrometry (APESI-MS). IAA was isolated by silica gel chromatography and HPLC. The effect on the growth of four marine microalgae of the pure IAA isolated from kelp extract was investigated. Exogenously added IAA from kelp enhanced the growth of Chlorella sp., Dunaliella salina and Porphyridium cruentum, but not that of Chaetoceros muelleri. IAA from kelp significantly inhibited the accumulation of soluble cellular proteins in Chlorella sp. and P. cruentum, and had a very significant effect on chlorophyll biosynthesis in Chlorella sp. However, there was no obvious effect of IAA on the regulation of biosynthesis of cellular polysaccharides in these four marine microalgae.     

Insight into glucosidase II from the red marine microalga Porphyridium sp. (Rhodophyta)

N‐glycosylation of proteins is one of the most important post‐translational modifications that occur in various organisms, and is of utmost importance for protein function, stability, secretion, and loca‐lization. Although the N‐linked glycosylation pathway of proteins has been extensively characterized in mammals and plants, not much information is available regarding the N‐glycosylation pathway in algae. We studied the α 1,3‐glucosidase glucosidase II (GANAB) glycoenzyme in a red marine microalga Porphyridium sp. (Rhodophyta) using bioinformatic and biochemical approaches. The GANAB‐gene was found to be highly conserved evolutionarily (compo‐sed of all the common features of α and β subunits) and to exhibit similar motifs consistent with that of homolog eukaryotes GANAB genes. Phylogenetic analysis revealed its wide distribution across an evolutionarily vast range of organisms; while the α subunit is highly conserved and its phylogenic tree is similar to the taxon evolutionary tree, the β subunit is less conserved and its pattern somewhat differs from the taxon tree. In addition, the activity of the red microalgal GANAB enzyme was studied, including functional and biochemical characterization using a bioassay, indicating that the enzyme is similar to other eukaryotes ortholog GANAB enzymes. A correlation between polysaccharide production and GANAB activity, indicating its involvement in polysaccharide biosynthesis, is also demonstrated. This study represents a valuable contribution toward understanding the N‐glycosylation and polysaccharide biosynthesis pathways in red microalgae.