1. Light and Energy. 2. Organization and Structure of Photosynthetic Systems. 3. History and Development of Photosynthesis. 4. Photosynthetic Pigments-Structure and Spectroscopy. 5. Antenna Complexes and Energy Transfer Processes. 6. Reaction Center Complexes. 7. Electron Transfer Pathways and Components. 8. Chemiosmotic Coupling and ATP Synthesis. 9. Carbon Metabolism. 10. Genetics, Assembly and Regulation of Photosynthetic. Systems. 11. Origin and Evolution of Photosynthesis. Appendix 1. Light, Energy and Kinetics

Molecular mechanisms of photosynthesis

Feeding the world population, 7.3 billion in 2015 and projected to increase to 9.5 billion by 2050, necessitates an increase in agricultural production of ~70% between 2005 and 2050. Soil degradation, characterized by decline in quality and decrease in ecosystem goods and services, is a major constraint to achieving the required increase in agricultural production. Soil is a non-renewable resource on human time scales with its vulnerability to degradation depending on complex interactions between processes, factors and causes occurring at a range of spatial and temporal scales. Among the major soil degradation processes are accelerated erosion, depletion of the soil organic carbon (SOC) pool and loss in biodiversity, loss of soil fertility and elemental imbalance, acidification and salinization. Soil degradation trends can be reversed by conversion to a restorative land use and adoption of recommended management practices. The strategy is to minimize soil erosion, create positive SOC and N budgets, enhance activity and species diversity of soil biota (micro, meso, and macro), and improve structural stability and pore geometry. Improving soil quality (i.e., increasing SOC pool, improving soil structure, enhancing soil fertility) can reduce risks of soil degradation (physical, chemical, biological and ecological) while improving the environment. Increasing the SOC pool to above the critical level (10 to 15 g/kg) is essential to set-in-motion the restorative trends. Site-specific techniques of restoring soil quality include conservation agriculture, integrated nutrient management, continuous vegetative cover such as residue mulch and cover cropping, and controlled grazing at appropriate stocking rates. The strategy is to produce “more from less” by reducing losses and increasing soil, water, and nutrient use efficiency.

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Restoring Soil Quality to Mitigate Soil Degradation

The unicellular green alga Chlamydomonas offers a simple life cycle, easy isolation of mutants, and a growing array of tools and techniques for molecular genetic studies. Among the principal areas of current investigation using this model system are flagellar structure and function, genetics of basal bodies (centrioles), chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. A genome project has begun with compilation of expressed sequence tag data and gene expression studies and will lead to a complete genome sequence. Resources available to the research community include wild-type and mutant strains, plasmid constructs for transformation studies, and a comprehensive on-line database.

Chlamydomonas as a model organism

Many species of microalgae have been used as source of nutrient rich food, feed and health promoting compounds. Among the commercially important microalgae, Haematococcus pluvialis is the richest source of natural astaxanthin which is considered as “super anti-oxidant". Natural astaxanthin produced by H. pluvialis has significantly greater antioxidant capacity than the synthetic one. Astaxanthin has important applications in the nutraceuticals, cosmetics, food, and aquaculture industries. Thanks to many researches it is now evident, that astaxanthin can significantly reduce free radicals and oxidative stress and help human body maintain a healthy state. With extraordinary potency and increase in demand, astaxanthin is one of the high-value microalgal products of the future. Thus, this comprehensive review summarizes the most important aspects of the biology, biochemical composition, biosynthesis and astaxanthin accumulation in the cells of H. pluvialis and its wide range of applications for humans and animals. In this paper, important and recent developments ranging from cultivation, harvest and postharvest bio-processing technologies to metabolic control and genetic engineering are reviewed in detail, focusing on biomass and astaxanthin production from this biotechnologically important microalga. Simultaneously, critical bottlenecks and major challenges in commercial scale production; current and prospective global market of H. pluvialis derived astaxanthin are also presented in a critical manner. A new biorefinery concept for H. pluvialis has been also suggested to guide towards economically sustainable approach for microalgae cultivation and processing. This report could serve as a useful guide to present current status of knowledge in the field and highlight key areas for future development of H. pluvialis astaxanthin technology and its large scale commercial implementation.

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Astaxanthin-Producing Green Microalga Haematococcus pluvialis: From Single Cell to High Value Commercial Products.

Chlamydomonas reinhardtii is a unicellular eukaryotic alga possessing a single chloroplast that is widely used as a model system for the study of photosynthetic processes. This report analyzes the surprising structural and evolutionary features of the completely sequenced 203,395-bp plastid chromosome. The genome is divided by 21.2-kb inverted repeats into two single-copy regions of ∼80 kb and contains only 99 genes, including a full complement of tRNAs and atypical genes encoding the RNA polymerase. A remarkable feature is that >20% of the genome is repetitive DNA: the majority of intergenic regions consist of numerous classes of short dispersed repeats (SDRs), which may have structural or evolutionary significance. Among other sequenced chlorophyte plastid genomes, only that of the green alga Chlorella vulgaris appears to share this feature. The program MultiPipMaker was used to compare the genic complement of Chlamydomonas with those of other chloroplast genomes and to scan the genomes for sequence similarities and repetitive DNAs. Among the results was evidence that the SDRs were not derived from extant coding sequences, although some SDRs may have arisen from other genomic fragments. Phylogenetic reconstruction of changes in plastid genome content revealed that an accelerated rate of gene loss also characterized the Chlamydomonas/Chlorella lineage, a phenomenon that might be independent of the proliferation of SDRs. Together, our results reveal a dynamic and unusual plastid genome whose existence in a model organism will allow its features to be tested functionally.

The Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats.

Nannochloropsis oceanica is an important source for omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) such as eicosapentaenoic acid (EPA, 20:5 n  − 3), and a potent candidate for biofuel production, due to its outstanding capability for rapid induction of triacylglycerol (TAG) overproduction In contrast to membrane lipids, TAG of N oceanica is poor in the valuable LC-PUFA, EPA We identified, cloned and characterized a N oceanica microsomal-like Δ12-desaturase ( NoD12 ) mediating the committing step of LC-PUFA biosynthesis by converting oleic acid (18:1 n  − 9) to linoleic acid (LA,18:2 n  − 6) We generated strains of N oceanica overexpressing NoD12 under the control of the stress-inducible endogenous lipid droplet surface protein (LDSP) promoter, resulting in robust expression under nitrogen starvation conditions The overexpression of NoD12 significantly altered fatty acid composition of total lipids and of individual lipid classes, such as a drastic increase in 18:2 proportion in phosphatidylcholine and in TAG was observed under nitrogen starvation Some LA was converted further toward LC-PUFA resulting in a substantial increase in arachidonic acid (20:4 n  − 6) in TAG Our data demonstrate the feasibility of metabolic engineering to increase LC-PUFA content in the biotechnologically important microalga using native genes and promoters, and provide novel insights into the regulation of LC-PUFA flux to TAG under nitrogen starvation

Metabolic engineering toward enhanced LC-PUFA biosynthesis in Nannochloropsis oceanica: Overexpression of endogenous Δ12 desaturase driven by stress-inducible promoter leads to enhanced deposition of polyunsaturated fatty acids in TAG

Cytoplasmic oil globules of Haematococcus pluvialis (Chlorophyceae) were isolated and analyzed for pigments, lipids and proteins. Astaxanthin appeared to be the only pigment deposited in the globules. Triacyglycerols were the main lipids (more than 90% of total fatty acids) in both the cell-free extract and in the oil globules. Lipid profile analysis of the oil globules showed that relative to the cell-free extract, they were enriched with extraplastidial lipids. A fatty acids profile revealed that the major fatty acids in the isolated globules were oleic acid (18:1) and linoleic acid (18:2). Protein extracts from the globules revealed seven enriched protein bands, all of which were possible globule-associated proteins. A major 33-kDa globule protein was partially sequenced by MS/MS analysis, and degenerate DNA primers were prepared and utilized to clone its encoding gene from cDNA extracted from cells grown in a nitrogen depleted medium under high light. The sequence of this 275-amino acid protein, termed the Haematococcus Oil Globule Protein (HOGP), revealed partial homology with a Chlamydomonas reinhardtii oil globule protein and with undefined proteins from other green algae. The HOGP transcript was barely detectable in vegetative cells, but its level increased by more than 100 fold within 12 h of exposure to nitrogen depletion/high light conditions, which induced oil accumulation. HOGP is the first oil-globule-associated protein to be identified in H. pluvialis, and it is a member of a novel gene family that may be unique to green microalgae.

Isolation of a Novel Oil Globule Protein from the Green Alga Haematococcus pluvialis (Chlorophyceae)

We have identified and isolated a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)1-like protein, from the diatom microalga Phaeodactylum tricornutum (PtDGAT1). The full-length cDNA sequences of PtDGAT1 transcripts revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from the single PtDGAT1 gene. PtDGAT1short encodes a 565 amino acid sequence that is homologous to several functionally characterized higher plant DGAT1 proteins, and 55% identical to the putative DGAT1 of the diatom Thalassiosira pseudonana, but shows little homology with other available putative and cloned algal DGAT sequences. PtDGAT1long lacks several catalytic domains, owing to a 63-bp nucleotide insertion in the mRNA containing a stop codon. Alternative splicing consisting of intron retention appears to regulate the amount of active DGAT1 produced, providing a possible molecular mechanism for increased triacylglycerol (TAG) biosynthesis in P. tricornutum under nitrogen starvation. DGAT mediates the last committed step in TAG biosynthesis, so we investigated the changes in expression levels of the two types of mRNA following nitrogen starvation inducing TAG accumulation. The abundance of both transcripts was markedly increased under nitrogen starvation, but much less so for PtDGAT1short. PtDGAT1 activity of PtDGAT1short was confirmed in a heterologous yeast transformation system by restoring DGAT activity in a Saccharomyces cerevisiae neutral lipid-deficient quadruple mutant strain (H1246), resulting in lipid body formation. Lipid body formation was only restored upon the expression of PtDGAT1short, and not of PtDGAT1long. The recombinant yeast appeared to display a preference for incorporating saturated C16 and C18 fatty acids into TAG.

Database
Nucleotide sequence data are available in the GenBank/EMBL/DDBJ databases under accession number HQ589265, sequence to be released November 15 2011.

Cloning and molecular characterization of a novel acyl‐CoA:diacylglycerol acyltransferase 1‐like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum

Are Thraustochytrids algae

An advanced shuttle-vector for efficient nuclear transformation and genetic engineering of Haematococcus pluvialis has been developed and tested by inserting linked trans-genes. The phytoene desaturase ( pds ) gene mutated in the codon for the amino acid at position 504 (from leucine to arginine), yields an enzyme resistant to the herbicide norflurazon and is used as an endogenous selection marker for transformation of H. pluvialis . A novel cloning vector allowing insertion of additional genes, both at the 5′ and the 3′ end of the mutated pds , has been designed by inserting the selection marker into the cloning vector pBluescript II SK(−) to give pBS– pds featuring the genomic mutated pds including 2000 bp of its promoter. In a second version pBS– pds short was created, by shortening the promoter sequence to 1000 bp. Unique restriction sites 5′ and 3′ of the selection marker have been reserved for insertion and simultaneous transformation with two transgenes. Transformation efficiency was significantly better than previously reported, achieving transformation frequencies of 2 × 10 − 5 both with long and short promoters, as well as with linear constructs. An expression cassette for the ble derived from vector pGenD- ble was inserted into pBS- pds either 5′ or 3′ of the pds , and successfully transformed into H. pluvialis , resulting in engineered strains weakly expressing the ble mRNA driven by the Chlamydomonas reinhardtii PsaD promoter. Both circular plasmids, as well as linear DNA fragments only consisting of the ble cassette fused to the pds selection marker were used successfully to engineer H. pluvialis . The plasmid constructs presented here, as well as the use of an endogenous dominant selection marker, represent a blueprint for the future successful production of safe, genetically modified microalgae, for the possible production of high value products or biofuels.

Stefan Leu

Papers

Metabolic engineering toward enhanced LC-PUFA biosynthesis in Nannochloropsis oceanica: Overexpression of endogenous Δ12 desaturase driven by stress-inducible promoter leads to enhanced deposition of polyunsaturated fatty acids in TAG

Isolation of a Novel Oil Globule Protein from the Green Alga Haematococcus pluvialis (Chlorophyceae)

Cloning and molecular characterization of a novel acyl‐CoA:diacylglycerol acyltransferase 1‐like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum

Are Thraustochytrids algae

Advanced methods for genetic engineering of Haematococcus pluvialis (Chlorophyceae, Volvocales)