Oil-accumulating microalgae have the potential to enable large-scale biodiesel production without competing for arable land or biodiverse natural landscapes. High lipid productivity of dominant, fast-growing algae is a major prerequisite for commercial production of microalgal oil-derived biodiesel. However, under optimal growth conditions, large amounts of algal biomass are produced, but with relatively low lipid contents, while species with high lipid contents are typically slow growing. Major advances in this area can be made through the induction of lipid biosynthesis, e.g., by environmental stresses. Lipids, in the form of triacylglycerides typically provide a storage function in the cell that enables microalgae to endure adverse environmental conditions. Essentially algal biomass and triacylglycerides compete for photosynthetic assimilate and a reprogramming of physiological pathways is required to stimulate lipid biosynthesis. There has been a wide range of studies carried out to identify and develop efficient lipid induction techniques in microalgae such as nutrients stress (e.g., nitrogen and/or phosphorus starvation), osmotic stress, radiation, pH, temperature, heavy metals and other chemicals. In addition, several genetic strategies for increased triacylglycerides production and inducibility are currently being developed. In this review, we discuss the potential of lipid induction techniques in microalgae and also their application at commercial scale for the production of biodiesel.

Microalgae have been widely reported as a promising source of biofuels, mainly based on their high areal productivity of biomass and lipids as triacylglycerides and the possibility for cultivation on non-arable land. The isolation and selection of suitable strains that are robust and display high growth and lipid accumulation rates is an important prerequisite for their successful cultivation as a bioenergy source, a process that can be compared to the initial selection and domestication of agricultural crops. We developed standard protocols for the isolation and cultivation for a range of marine and brackish microalgae. By comparing growth rates and lipid productivity, we assessed the potential of subtropical coastal and brackish microalgae for the production of biodiesel and other oil-based bioproducts. This study identified Nannochloropsis sp., Dunaniella salina and new isolates of Chlorella sp. and Tetraselmis sp. as suitable candidates for a multiple-product algae crop. We conclude that subtropical coastal microalgae display a variety of fatty acid profiles that offer a wide scope for several oil-based bioproducts, including biodiesel and omega-3 fatty acids. A biorefinery approach for microalgae would make economical production more feasible but challenges remain for efficient harvesting and extraction processes for some species.

Linear gramicidin is a membrane channel forming pentadecapeptide that is produced via the nonribosomal pathway. It consists of 15 hydrophobic amino acids with alternating L-and D-configuration forming a ␤-helix-like structure. It has an N-formylated valine and a C-terminal ethanolamine. Here we report cloning and sequencing of the entire biosynthetic gene cluster as well as initial biochemical analysis of a new reductase domain. The biosynthetic gene cluster was identified on two nonoverlapping fosmids and a 13-kilobase pair (kbp) interbridge fragment covering a region of 74 kbp.Four very large open reading frames, lgrA, lgrB, lgrC, and lgrD with 6.8, 15.5, 23.3, and 15.3 kbp, were identified and shown to encode nonribosomal peptide synthetases with two, four, six, and four modules, respectively. Within the 16 modules identified, seven epimerization domains in alternating positions were detected as well as a putative formylation domain fused to the first module LgrA and a putative reductase domain attached to the C-terminal module of LgrD. Analysis of the substrate specificity by phylogenetic studies using the residues of the substrate-binding pockets of all 16 adenylation domains revealed a good agreement of the substrate amino acids predicted with the sequence of linear gramicidin. Additional biochemical analysis of the three adenylation domains of modules 1, 2, and 3 confirmed the colinearity of this nonribosomal peptide synthetase assembly line. Module 16 was predicted to activate glycine, which would then, being the C-terminal residue of the peptide chain, be reduced by the adjacent reductase domain to give ethanolamine, thereby releasing the final product N-formyl-pentadecapeptideethanolamine. However, initial biochemical analysis of this reductase showed only a one-step reduction yielding the corresponding aldehyde in vitro.Gramicidin is a pentadecapeptide antibiotic produced by Bacillus brevis ATCC 8185 during its sporulation phase (1). The primary structure of gramicidin A was determined as formyl-

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent serine endopeptidases found in almost every organism. Database searches revealed that 16 Deg paralogues are encoded by the genome of Arabidopsis thaliana, six of which were experimentally shown to be located in chloroplasts, one in peroxisomes, one in mitochondria and one in the nucleus. Two more Deg proteases are predicted to reside in chloroplasts, five in mitochondria (one of them with a dual chloroplastidial/mitochondrial localization) and the subcellular location of one protein is uncertain. This review summarizes the current knowledge on the role of Deg proteases in maintaining protein homeostasis and protein processing in various subcompartments of the plant cell. The chloroplast Deg proteases are the best examined so far, especially with respect to their role in the degradation of photodamaged photosynthetic proteins and in biogenesis of photosystem II (PSII). A combined action of thylakoid lumen and stroma Deg proteases in the primary cleavage of photodamaged D1 protein from PSII reaction centre is discussed on the basis of a recently resolved crystal structure of plant Deg1. The peroxisomal Deg protease is a processing enzyme responsible for the cleavage of N-terminal peroxisomal targeting signals (PTSs). A. thaliana mutants lacking this enzyme show reduced peroxisomal β-oxidation, indicating for the first time the impact of protein processing on peroxisomal functions in plants. Much less data is available for mitochondrial and nuclear Deg proteases. Based on the available expression data we hypothesize a role in general protein quality control and during acquired heat resistance.

Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. Recently, candidates for the responsible processing protease were identified from plants (DEG15) and mammals (TYSND1). We demonstrate that plants lacking DEG15 show an expressed phenotype potentially linked to reduced b-oxidation, indicating the impact of protein processing on peroxisomal functions in higher eukaryotes. Mutational analysis of Arabidopsis (Arabidopsis thaliana) DEG15 revealed that conserved histidine, aspartic acid, and serine residues are essential for the proteolytic activity of this enzyme in vitro. This indicates that DEG15 and related enzymes are trypsin-like serine endopeptidases. Deletion of a plant-specific stretch present in the protease domain diminished, but did not abolish, the proteolytic activity of DEG15 against the PTS2-containing glyoxysomal malate dehydrogenase. Fluorescence microscopy showed that a DEG15-green fluorescent protein fusion construct is targeted to peroxisomes in planta. In vivo studies with isolated homozygous deg15 knockout mutants and complemented mutant lines suggest that this enzyme mediates general processing of PTS2-containing proteins.

The family of Deg proteases is present in nearly all organisms from bacteria to higher plants. This family consists of ATP‐independent serine endopeptidases with a catalytic domain of trypsin type and up to three PDZ domains, involved in protein–protein interactions. Sixteen deg genes (originally named degP1–16) were found in Arabidopsis thaliana, and the chloroplast location was predicted or experimentally proven for seven proteins. The cyanobacterium Synechocystis sp. PCC6803 contains three Deg homologues, HtrA (DegP), HhoA (DegQ) and HhoB (DegS), but their number can vary between one and six in other photosynthetic Prokaryota. Interestingly, all of these proteases are evolutionarily more closely related within one species than proteases with the same names present in other organisms. This means that Deg proteases from A. thaliana are not necessarily the closest relatives of cyanobacterial DegP. Therefore, we propose to change the misleading original name ‘DegP’ to ‘Deg’ for A. thaliana enzymes. Here, we summarize the expression, location and functions of Deg proteases from cyanobacteria and chloroplasts of higher plants, with special emphasis on their role in the photosystem II (PSII) repair cycle under light stress conditions.

In plants exposed to high irradiances of visible light, the D1 protein in the reaction center of photosystem II is oxidatively damaged and rapidly degraded. Earlier work in our laboratory showed that the serine protease Deg2 performs the primary cleavage of photodamaged D1 protein in vitro. Here, we demonstrate that the rate of D1 protein degradation under light stress conditions in Arabidopsis mutants lacking the Deg2 protease is similar to those in wild-type plants. Therefore, we propose that several redundant D1 protein degradation pathways might exist in vivo.

BackgroundThe Deg/HtrA family of ATP-independent serine endopeptidases is present in nearly all organisms from bacteria to human and vascular plants. In recent years, multiple deg/htrA protease genes were identified in various plant genomes. During genome annotations most proteases were named according to the order of discovery, hence the same names were sometimes given to different types of Deg/HtrA enzymes in different plant species. This can easily lead to false inference of individual protease functions based solely on a shared name. Therefore, the existing names and classification of these proteolytic enzymes does not meet our current needs and a phylogeny-based standardized nomenclature is required.ResultsUsing phylogenetic and domain arrangement analysis, we improved the nomenclature of the Deg/HtrA protease family, standardized protease names based on their well-established nomenclature in Arabidopsis thaliana, and clarified the evolutionary relationship between orthologous enzymes from various photosynthetic organisms across several divergent systematic groups, including dicots, a monocot, a moss and a green alga. Furthermore, we identified a “core set” of eight proteases shared by all organisms examined here that might provide all the proteolytic potential of Deg/HtrA proteases necessary for a hypothetical plant cell.ConclusionsIn our proposed nomenclature, the evolutionarily closest orthologs have the same protease name, simplifying scientific communication when comparing different plant species and allowing for more reliable inference of protease functions. Further, we proposed that the high number of Deg/HtrA proteases in plants is mainly due to gene duplications unique to the respective organism.