Biorefineries enable the circular, sustainable, and economic use of waste resources if value-added products can be recovered from all the generated fractions at a large-scale. In the present studies the comparison and assessment for the production of value-added compounds (e.g., proteins, lutein, and lipids) by the microalga Chlorella sorokiniana grown under photoautotrophic or heterotrophic conditions was performed. Photoautotrophic cultivation generated little biomass and lipids, but abundant proteins (416.66 mg/g_CDW) and lutein (6.40 mg/g_CDW). Heterotrophic conditions using spruce hydrolysate as a carbon source favored biomass (8.71 g/L at C/N 20 and 8.28 g/L at C/N 60) and lipid synthesis (2.79 g/L at C/N 20 and 3.61 g/L at C/N 60) after 72 h of cultivation. Therefore, heterotrophic cultivation of microalgae using spruce hydrolysate instead of glucose offers a suitable biorefinery concept at large-scale for biodiesel-grade lipids production, whereas photoautotrophic bioreactors are recommended for sustainable protein and lutein biosynthesis.

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.

Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is higher globally than production due to increased incidences of various heart conditions. The main source of streptokinase is various strains of Streptococci. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD gene-mediated post-translational modification of streptolysin, an inhibitor of streptokinase expression through the degradation of FasX small RNA (through CoV/RS), which stabilizes streptokinase mRNA. In order to improve the stability of mRNA and increase the expression of streptokinase, which is inhibited by SagA, we used CRISPR-Cas9 to successfully knockout the SagD gene and observed a 13.58-fold increased expression of streptokinase at the transcript level and 1.48-fold higher expression at the protein level in the mutant strain compared to wild type. We have demonstrated the successful gene knockout of SagD using CRISPR-Cas9 in S. equisimilis, where an engineered strain can be further used for overexpression of streptokinase for therapeutic applications.