Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum.

There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.

High-efficiency promoter-driven coordinated regulation of multiple metabolic nodes elevates lipid accumulation in the model microalga Phaeodactylum tricornutum.

BACKGROUND: Microalgal metabolic engineering holds great promise for the overproduction of a wide range of commercial bioproducts. It demands simultaneous manipulation of multiple metabolic nodes. However, high-efficiency promoters have been lacking. RESULTS: Here we report a strong constitutive promoter Pt211 in expressing multiple target genes in oleaginous microalga Phaeodactylum tricornutum. Pt211 was revealed to contain significant cis-acting elements. GUS reporter and principal genes glycerol-3-phosphate acyltransferase (GPAT) and diacylglycerol acyltransferase 2 (DGAT2) involved in triacylglycerol biosynthesis were tested under driven of Pt211 in P. tricornutum. GUS staining and qPCR analysis showed strong GUS expression. DGAT2 and GPAT linked with a designed 2A sequence exhibited higher transcript abundances than WT, while algal growth and photosynthesis were not impaired. CONCLUSION: The total lipid content increased notably by 2.6-fold compared to WT and reached up to 57.5% (dry cell weight). Overall, our findings report a strong promoter and a strategy for coordinated manipulation of complex metabolic pathways.

Mechanistic insights into the effects of diuron exposure on Alexandrium pacificum.

Diuron (N-(3,4-dichlorophenyl)-N,N­dimethylurea, DCMU), a ureic herbicide, is extensively used in agriculture to boost crop productivity; however, its extensive application culminates in notable environmental pollution, especially in aquatic habitats. Therefore, the present study investigated the effect of diuron on the dinoflagellate Alexandrium pacificum, which is known to induce harmful algal blooms (HAB), and its potential to biodegrade DCMU. Following a four-day DCMU exposure, our results revealed that A. pacificum proficiently assimilated DCMU at concentrations of 0.05 mg/L and 0.1 mg/L in seawater, attaining a complete reduction (100 % efficiency) after 96 h for both concentrations. Moreover, evaluations of paralytic shellfish toxins content indicated that cells subjected to higher DCMU concentrations (0.1 mg/L) exhibited reductions of 73.4 %, 86.7 %, and 75 % in GTX1, GTX4, and NEO, respectively. Exposure to DCMU led to a notable decrease in A. pacificum's photosynthetic efficacy, accompanied by increased levels of reactive oxygen species (ROS) and suppressed cell growth, with a growth inhibition rate of 41.1 % at 72 h. Proteomic investigations pinpointed the diminished expression levels of specific proteins like SxtV and SxtW, linked to paralytic shellfish toxins (PSTs) synthesis, as well as key proteins associated with Photosystem II, namely PsbA, PsbD, PsbO, and PsbU. Conversely, proteins central to the cysteine biosynthesis pathways exhibited enhanced expression. In summary, our results preliminarily resolved the molecular mechanisms underlying the response of A. pacificum to DCMU and revealed that DCMU affected the synthesis of PSTs. Meanwhile, our data suggested that A. pacificum has great potential in scavenging DCMU.

Bioremediation of Catechol and Concurrent Accumulation of Biocompounds by the Microalga Crypthecodinium cohnii.

Burgeoning commercial applications of catechol have led to its excessive accumulation in the environment, thereby posing a severe ecological threat. Bioremediation has emerged as a promising solution. The potential of the microalga Crypthecodinium cohnii to degrade catechol and use the byproduct as a carbon source was investigated in this study. Catechol significantly increased C. cohnii growth and was rapidly catabolized within 60 h of cultivation. Transcriptomic analysis highlighted the key genes involved in catechol degradation. Real-time polymerase chain reaction (RT-PCR) analysis showed that transcription of key genes CatA, CatB, and SaID involved in the ortho-cleavage pathway was remarkably increased by 2.9-, 4.2-, and 2.4- fold, respectively. Key primary metabolite content was also markedly altered, with a specific increment in polyunsaturated fatty acids. Electron microscopy and antioxidant analysis showed that C. cohnii could tolerate catechol treatment without morphological aberrations or oxidative stress. The findings provide a strategy for C. cohnii in the bioremediation of catechol and concurrent polyunsaturated fatty acids (PUFA) accumulation.

Transcriptional regulation of microalgae for concurrent lipid overproduction and secretion.

Commercialization of algal lipids and biofuels is still impractical owing to the unavailability of lipogenic strains and lack of economically viable oil extraction strategies. Because lipogenesis is governed by multiple factors, success in generating industrial-suitable algal strains using conventional strategies has been limited. We report the discovery of a novel bZIP1 transcription factor, NobZIP1, whose overexpression results in a remarkable elevation of lipid accumulation and lipid secretion in a model microalga Nannochloropsis oceanica, without impairing other physiological properties. Chromatin immunoprecipitation-quantitative PCR analysis revealed that the key genes up- and down-regulated by NobZIP1 are involved in lipogenesis and cell wall polymer synthesis, respectively, which, in turn, induce lipid overproduction and secretion. Among these regulated genes, UDP-glucose dehydrogenase was shown to alter cell wall composition, thus also boosting lipid secretion. In summary, these results offer a comprehensive strategy for concurrent lipid overproduction and secretion, strongly increasing the commercial potential of microalgae.