The regulation of genes related to lipid metabolism by genetic engineering is an important way to increase the accumulation of lipids in microalgae. DNA binding with one finger (DOF) is a plant-specific transcription factor in higher plants, where it regulates carbon and nitrogen metabolic pathways by regulating key genes involved in these pathways. Overexpression of DOF can increase lipid production in plants; however, it is not clear whether overexpression of DOF can increase lipids in microalgae. In this study, we cloned a DOF transcription factor, crDOF, from Chlamydomonas reinhardtii. The sequence of this transcription factor is 1875 bp and encodes a peptide of 624 amino acids with a conserved DOF domain. Overexpression of crDOF in C. reinhardtii significantly increased the intracellular lipid content. The content of total fatty acids in the transgenic algae line Tranc-crDOF-12 was 126.01 μg/mg (dry weight), which was 23.24% higher than that of the wild type. Additionally, the content of unsaturated fatty acids in the transgenic Tranc-crDOF-12 line increased significantly. Fluorescence quantitative PCR analysis showed that in the transgenic line Tranc-crDOF-12, the expression levels of BCC1, FAT1, SQD1, MGD1, DGD1 and PGP1 genes were significantly upregulated, while the expression levels of ACP1, ACS1, CIS1 and SQD2 were downregulated. Our results confirm that crDOF increases intracellular lipids in C. reinhardtii by regulating key genes involved in lipid metabolism. According to these findings, we propose that enhancing the lipid content in microalgae by overexpressing DOF may be achieved in other industrial strains of microalgae and be employed for the industrial production of biodiesel.

Eukaryotic green microalgae show considerable promise for the sustainable light-driven biosynthesis of high-value fine chemicals, especially terpenoids because of their fast and inexpensive phototrophic growth. Here, the novel isopentenol utilization pathway (IUP) was introduced into Chlamydomonas reinhardtii to enhance the hemiterpene (isopentenyl pyrophosphate, IPP) titers. Then, diphosphate isomerase (IDI) and limonene synthase (MsLS) were further inserted for limonene production. Transgenic algae showed 8.6-fold increase in IPP compared with the wild type, and 23-fold increase in limonene production compared with a single MsLS expressing strain. Following the culture optimization, the highest limonene production reached 117 µg/L, when the strain was cultured in a opt2 medium supplemented with 10 mM isoprenol under a light: dark regimen. This demonstrates that transgenic algae expressing the IUP represent an ideal chassis for the high-value terpenoid production. The IUP will facilitate further the metabolic and enzyme engineering to enhance the terpenoid titers by significantly reducing the number of enzyme steps required for an optimal biosynthesis.

Microalgae biofuel has become the most promising renewable energy over the past few years. But limitations still exist because of its high cost. Although, efforts have been made in enhancement of lipid productivity, the major cost problem in harvesting and oil extraction is still intractable. Thus, the idea of fatty acids (FAs) secretion which can massively facilitate algae harvesting and oil extraction was investigated here. The cDNAs of two long-chain acyl-CoA synthetases (LACSs) genes were cloned from Chlamydomonas reinhardtii and named as cracs1 and cracs2. They showed different substrate adaptation in the yeast complementation experiments. Cracs2 could utilize FAs C12:0, C14:0, C16:0, C18:0, C16:1 and C18:1, while crac1 could only utilize substrate C14:0, C16:1 and C18:1. Knockdown of cracs1 and cracs2 in C. reinhardtii resulted in accumulation of intracellular lipids. The total intracellular lipids contents of transgenic algae q-15 (knockdown of cracs1) and p-13 (knockdown of cracs2) were 45 and 55 %, respectively higher than that of cc849. Furthermore, FAs secretion was discovered in both transgenic algae. Secreted FAs can reach 8.19 and 9.66 mg/109 cells in q-15 and p-13, respectively. These results demonstrated the possibility of FAs secretion by microalgae and may give a new strategy of low-cost oil extraction. According to our findings, we proposed that FAs secretion may also be achieved in other species besides Chlamydomonas reinhardtii by knocking-down cracs genes, which may promote the future industrial application of microalgae biofuels.

Microalgae are promising microorganisms used to produce value-added products or to develop sustainable approaches for environmental remediation. The ATP-binding cassette proteins (ABCs) of Chlamydomonas reinhardtii have been characterized as indispensable transporters for CO₂ concentrating mechanism, lipid biosynthesis, and heavy metal sequestration. However, few microalgal ABC proteins have been studied compared with higher plants or non-photosynthetic microorganisms. This study performed a genome-wide, evolutionary, and transcriptomic survey of C. reinhardtii ABC proteins (CrABCs). A total of 75 CrABCs were identified and classed into eight ABC subfamilies, from ABCA to ABCI. We found that no whole or partial genome duplication events occurred in C. reinhardtii after the ancient endosymbiosis events, but gene duplications occurred in a small range of chromosomal regions, which forced ABC family expansion. Abundant light, abscisic acid, and jasmonic acid response cis-elements were mapped in the CrABC promoters, coinciding with the evolutionary history of hormone signaling in Chlorophyta. The expression survey under light/dark rhythms revealed a close bond of CrABCs with cell division and development. A broad study of CrABCs supported their expected roles in heavy metal detoxification, lipid metabolism, and environmental adaptation. Moreover, the evolutionary and expression survey predicted the functions of unknown CrABCs, which are elaborated in the text. Two half-size CrABCGs—CrABCG3 and CrABCG26—were described as plasma-membrane transporters that might participate in lipidic compound secretion. This study provides fundamental and exhaustive information about CrABCs, which are indispensable for the functional elucidation of ABC proteins in microalgae.

Microalgal lipids are essential for biofuel and dietary supplement production. Lipid engineering for higher production has been studied for years. However, due to the complexity of lipid metabolism, single-gene engineering gradually encounters bottlenecks. Multiple gene regulation is more beneficial to boosting lipid accumulation and further clarifying the complex regulatory mechanism of lipid biosynthesis in the homeostasis of lipids, carbohydrates, and protein metabolism. Here, three lipid-related genes, DOF, LACS2, and CIS, were co-regulated in Chlamydomonas reinhartii by two circles of transformation to overexpress DOF and knock down LACS2 and CIS simultaneously. With the multiple regulations of these genes, the intracellular lipids and FA content increased by 142% and 52%, respectively, compared with CC849, whereas the starch and protein contents decreased by 45% and 24%. Transcriptomic analysis showed that genes in TAG and FA biosynthesis were up-regulated, and genes in starch and protein metabolism were down-regulated. This revealed that more carbon precursor fluxes from starch and protein metabolism were redirected towards lipid synthesis pathways. These results showed that regulating genes in various metabolisms contributed to carbon flux redirection and significantly improved intracellular lipids, demonstrating the potential of multiple gene regulation strategies and providing possible candidates for lipid overproduction in microalgae.

Lipase from Aspergillus niger was “doubly immobilized” with SiO₂ nanoparticles in sol-gel powders prepared via the base-catalyzed polymerization of tetramethoxysilane (TMOS) and methyltreimethoxysilane (MTMS). The hydrolytic activity of the immobilized lipase was measured using the p-nitrophenyl palmitate hydrolysis method. The results showed that the optimum preparation conditions for the gels were made using a MTMS/TMOS molar ratio of 5, 60 mg of SiO₂ nanoparticles, a water/silane molar ratio of 12, 120 mg of enzyme supply, and 120 μL of PEG400. Under the optimal conditions, the immobilized lipase retained 92% of the loading protein and 94% of the total enzyme activity. Characteristic tests indicated that the immobilized lipase exhibited much higher thermal and pH stability than its free form, which shows great potential for industrial applications.