Comprehensive sequence and structure analysis of algal lipid catabolic enzyme Triacylglycerol lipase: an in silico study to vitalize the development of optimum engineered strains with high lipid productivity.

Microalgae as an alternative renewable resource for biofuel production have captured much significance. Nonetheless, its economic viability is a field of major concern for researchers. Unraveling the lipid catabolic pathway and gaining insights into the sequence-structural features of its primary functioning enzyme, Triacylglycerol lipase, will impart valuable information to target microalgae for augmented lipid content. In the present study, a genome-wide comparative study on putative Triacylglycerol lipase (TAGL) enzyme from algal species belonging to varied phylogenetic lineages was performed. The comprehensive sequence analysis revealed that TAGL comprises of three distinct conserved domains, such as, Patatin, Class III Lipase, and Abhydro_lipase, and also confirmed the ubiquitous presence of GXSXG motif in the sequences analyzed. In the absence of a crystal structure of algal TAGL till date, we developed the first 3D model of patatin domain of TAGL from an oleaginous microalga, Phaedactylum tricornutum, employing homology modeling, docking and molecular dynamic simulations methods. The domain-substrate complex having the low-ranking docking score revealed the binding of palmitic acid to the TAGL patatin domain surface with strong hydrogen bond interactions. The simulation results implied that the substrate-complexed patatin domain and the free enzyme adopted a more stable conformation after 40 ns. This is the first ever attempt to provide in-silico insights into the structural and dynamical insights on catalytic mechanism of the TAGL patatin domain. Subsequently, these findings aided our understanding on their structural stability, folding mechanism and protein-substrate interactions, which could be further utilized to design site-specific mutagenic experiments for engineering microalgal strains.Communicated by Ramaswamy H. Sarma.

Genome-based identification and comparative analysis of enzymes for carotenoid biosynthesis in microalgae.

Microalgae are potential feedstocks for the commercial production of carotenoids, however, the metabolic pathways for carotenoid biosynthesis across algal lineage are largely unexplored. This work is the first to provide a comprehensive survey of genes and enzymes associated with the less studied methylerythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway as well as the carotenoid biosynthetic pathway in microalgae through bioinformatics and comparative genomics approach. Candidate genes/enzymes were subsequently analyzed across 22 microalgae species of lineages Chlorophyta, Rhodophyta, Heterokonta, Haptophyta, Cryptophyta, and known Arabidopsis homologs in order to study the evolutional divergence in terms of sequence-structure properties. A total of 403 enzymes playing a vital role in carotene, lutein, zeaxanthin, violaxanthin, canthaxanthin, and astaxanthin were unraveled. Of these, 85 were hypothetical proteins whose biological roles are not yet experimentally characterized. Putative functions to these hypothetical proteins were successfully assigned through a comprehensive investigation of the protein family, motifs, intrinsic physicochemical features, subcellular localization, pathway analysis, etc. Furthermore, these enzymes were categorized into major classes as per the conserved domain and gene ontology. Functional signature sequences were also identified which were observed conserved across microalgal genomes. Additionally, the structural modeling and active site architecture of three vital enzymes, DXR, PSY, and ZDS catalyzing the vital rate-limiting steps in Dunaliella salina were achieved. The enzymes were confirmed to be stereochemically reliable and stable as revealed during molecular dynamics simulation of 100 ns. The detailed functional information about individual vital enzymes will certainly help to design genetically modified algal strains with enhanced carotenoid contents.

dEMBF v2.0: An Updated Database of Enzymes for Microalgal Biofuel Feedstock.

In light of increasing algal genomics data and knowledge of biosynthetic pathways responsible for biofuel production, an integrated resource for easy access to all information is essential to improve our understanding of algal lipid metabolism. Against this backdrop, dEMBF v2.0, a significantly updated and improved version of our database of microalgae lipid biosynthetic enzymes for biofuel production, has been developed. dEMBF v2.0 now contains a comprehensive annotation of 2018 sequences encoding 35 enzymes, an increase of over 7-fold as compared with the first version. Other improved features include an increase in species coverage to 32 algal genomes, analysis of additional metabolic pathways, expanded annotation thoroughly detailing sequence and structural features, including enzyme-ligand interactions, and integration of supporting experimental evidence to demonstrate the role of enzymes in increasing lipid content. Along with a complete redesign of the interface, the updated database provides several inbuilt tools and user-friendly functionalities for more interactive and dynamic visualization of data.