Molecular insight into cellulose degradation by the phototrophic green alga Scenedesmus

BARCHIESI, JULIETA

Rosario

Congreso; LIX Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2023

On the other hand, growth conditions were achieved in which Scenedesmus expresses a single cellulolytic enzyme in the supernatant. It was purified, concentrated and its enzymatic activity was tested against different substrates. The term cellulolytic activity includes β-endoglucanase, exocellulase and β-glucosidase (or cellobiase) enzymes, and they can be identified using different substrates and analyzing the reaction product generated in each case. When using CMC as a substrate, no release of disaccharides was detected as a reaction product, or by quantifying reducing ends with DNS. Suggesting that the supernatant enzyme does not present significant exocellulase activity. On the other hand, when its β-glucosidase activity was assayed using cellobiose as a substrate, it was significant and we obtained its kinetic parameters. This result evidences that cellobiase activity is predominant in supernatant in the assayed conditions.Then, optimum temperature and pH of β-glucosidase activity were determined. The thermal stability of the enzyme was tested, which presented stability up to temperatures close to 50°C. Above which it maintains a residual activity close to 77% and 60% at 60°C and 70°C, respectively.Additionally, we corroborated that the β -glucosidase activity was strongly repressed by the presence of the reaction product (glucose); and we analyze the effect of different metal ions, solvents and detergents previously reported as activators or repressors of β-glucosidase activity and usually present in industrial saccharification processes.Later, enzyme production was analyzed in a liquid medium with CMC, in order to identify the cellulases that make up the Scenedesmus secretome. The supernatant of the cultures grown for 5 days in liquid media supplemented with CMC was precipitated with NH4SO2 and native polyacrylamide gels containing CMC were resolved. Subsequently, the gels were revealed by staining with Congo red. The presence of hydrolysis bands was observed in the region corresponding to the protein bands of 100 KDa. They were cut from an identical gel, without CMC and sent to the Mass Spectrometry services of the Center for Chemical and Biological Studies (CEQUIBIEM) and the Institute of Cellular and Molecular Biology of Rosario (IBR) for identification. Once the peptide sequences were obtained, they were analyzed with different databases, including Uniprot, NCBI and Phycocosm.We have not yet identified full enzymatic sequences, only partial ones when comparing with existing microalgae proteomic or genomic database. Therefore, we have sent genomic DNA and cDNA samples of our S. quadricauda SFE-03 strain to be sequenced by Whole Genome Sequencing (WGS, Illumina) to Novogene USA. High data quality was obtained for both sequences (Effectiveness: 99.84% for genomic DNA and 99.85% for cDNA). We have recently finished it assembly and we still analyzing it.In parallel, with the objective of analyze the evolutionary conservation of the cellulolytic enzymes in Scenedemaceae, we carried out a bioinformatic analysis of the Scenedesmus public genome sequences.We detected the presence of extracellular endoglucanases, exocellulases and beta-glucosidases in the genomes of the five species analyzed (Scenedesmus obliquus EN0004 v1.0, Scenedesmus obliquus UTEX B 3031, Scenedesmus obliquus var. DOE0013 v1.0, Scenedesmus sp. NREL 46B-D3 v1.0, and S. quadricauda LWG002611). The comparison of sequences of these cellulases with hydrolytic enzymes from other organisms by means of multisequence alignments and phylogenetic trees showed that they belong to the families of glycosylhydrolases 1, 5, 9 and 10 (GH1, GH5, GH9 and GH10). Using the InterproScan program, we detected the presence of cellulose-binding domains, conserved in several of the putative endoglucanases analyzed from the GH5 and GH9 families. In addition, most of them showed greater sequence similarity with enzymes frominvertebrates, fungi, bacteria, and other microalgae than with plant cellulases. The obtained 3D modeling data showed that both the main structures of the modeled proteins and the amino acid residues involved in catalysis and substrate binding are well conserved in Scenedesmus enzymes. These results have recently been published in PROTEINS: Structure, Function, and Bioinformatics. (Velazquez et al 2023, “Molecular insight into cellulose degradation by the phototrophic green alga Scenedesmus.”)This ability to degrade exogenous cellulose probably evolved to take advantage of the mother cell walls released and accumulated in the media during cell division (autolysis). This function would currently be maintained in obligate autotroph algae, while in mixotrophic algae such as Scenedesmus, it would allow the use of the cellulose as an alternative source of carbon under certain conditions. Scenedesmus is usually found in freshwater rivers and lakes, but also in brackish habitats. Algae cells may be buried in sludge or exposed to low light surroundings deep in the river, but sometimes they could return to the high light environment of the water column. Having the ability of doing both photosynthesis or assimilating polysaccharides allows these cells to respond promptly to the new source of energy and light. This strategy would represent an advantage to certain species that would have allowed Scenedesmaceae to occupy many environments in nature.These findings open the opportunity to identify new cellulases from algae, as well as to perform their functional characterization to evaluate the possibility of their use in various biotechnological applications.Another of our research projects implies the obtention of transgenic algae with a modified cell wall. A strong barrier to the use of the components produced by microalgae is the high resistance to enzymatic and chemical hydrolysis of its cell walls. Scenedemus exhibit a trilaminar cell wall structure, with an outer layer formed mainly by pectins, and their inner layers of cellulose and insoluble algenans.The generation of transgenic microalgae with modifications at the cell wall level is a biotechnological approach with promising results.Numerous enzymes and proteins are involved in the synthesis, degradation and remodeling of the plant cell wall. A common aspect of the structure of most of them is their modular organization; which includes a characteristic catalytic domain of each enzyme and one or more carbohydrate binding domains (CBM) linked by a loosely structured chain.In our laboratory we have characterized several starch binding domains (SBDs) from A. thaliana. These domains are highly conserved in plants and algae. Our group demonstrated that cell wall-directed expression of SBDs in Arabidopsis thaliana decreases its total cell wall content in all organs and a significant increase in the levels of hemicelluloses and stem pectins. In this context, we transformed Scenedesmus cells with a GP-SBD chimeric construct, where this module is directed towards its cell wall using a Chlamydomonas signal peptide.The analysis of the transgenic lines obtained showed that the modified cells are larger than those of the wild type, their surface/volume ratio is modified and favors their flocculation. The components of the cell wall are altered, specifically the pectin layer, increasing the union between cells and forming clusters. No significant effect was observed on the adsorption of metals from the medium, but on the composition of soluble metabolites, evidencing an increase in the levels of nitrogen-rich amino acids. We will continue characterizing these lines, but the phenotypes already identifiedconfirm the usefulness of using SBD targeting to modify cell walls for different biotechnological purposes in microalgae.