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The unicellular green microalga Desmodesmus sp. S1 can produce total lipid over 50% of cell dry weight. A mutant D90G-19 induced by 12C6+ heavy ion irradiation generates more lipid which is 20% higher than its wild type (WT) under high light. The gene expression profiles in the photosynthetic pathway between the Desmodesmus sp. S1 WT and D90G-19 were compared, and several key genes were verified using qRT-PCR and western blotting. The psbA and psbD encoding the D1 and D2 subunits of PSII in D90G-19 were upregulated and higher than WT under high light, while an LHCB gene of Desmodesmus sp. was downregulated dramatically. The expression level of LHCB of D90G-19 was less than that of WT. The improved photoprotection capacity of D90G-19 was attributed to LHCII antennae probably due to the mutation of LHCB gene, which led to less photon into the electron transport chain. Furthermore, LHCBM, a homologous gene of LHCB in model microalgae N. gaditana Cas9+ strain, was knocked out using CRISPR/Cas9. The resulting mutant NgCas9-LHCBM showed an improved capacity to protect the photosynthetic complexes by dissipating extra light energy through non-photochemical quenching pathway. Therefore, the photoprotection capacity of microalgae can be improved by engineering the quantity of PSII subunits to fine-tune PSII reaction centers and the adjacent LHCII antennae.

Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite -4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases.

Large-scale production of biofuels and other high-value products from microalgae focuses on strain selection with desired characteristics such as high lipid productivity and robustness. However, microalgae often suffer severe biomass losses due to contamination that comes in the form of herbivores, especially rotifers. In this study, we found that the total lipid content of 54 microalgal strains from Eastern China ranged from 12.6 to 59.9% of dry cell weight (dw), with an average of 35.4% dw. Lipid productivity of these microalgal strains varied from 0.04 to 0.14 g L−1 day−1, and 15 showed lipid production rates over 0.10 g L−1 day−1. The fatty acid profiles of 34 microalgal strains with lipid content over 40% dw were analyzed by gas chromatography. We found that the amount of C18:1 increased with increasing total lipid content, whereas C14:0 and C16:0 showed a negative correlation to the total amount of lipids. An experimental rotifer-microalgae predator-prey model was constructed, and the anti-rotifer robustness of these microalgal strains was evaluated. We found that Didymogenes sp. HN-4 can depress the growth of rotifers significantly. This study demonstrates the biodiversity of the native Chinese microalgae in lipid productivity and robustness, which lays the groundwork for mass algal culture.