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Antisense knockdown of pyruvate dehydrogenase kinase promotes the neutral lipid accumulation in the diatom Phaeodactylum tricornutum.

ABSTRACT: BACKGROUND: Microalgae have been an emerging biofuel resource; however, the germplasm improvement has been slow due to the lack of molecular tools. Pyruvate dehydrogenase kinase (PDK) deactivates the pyruvate dehydrogenase complex (PDC) which catalyzes the oxidative decarboxylation of pyruvate. Acetyl-CoA production via PDC is important in plant tissues that are active in fatty acid synthesis. RESULTS: A 1261-bp cDNA of a putative PDK gene (PtPDK) was cloned from a diatom Phaeodactylum tricornutum, and PtPDK antisense knockdown transgenic diatoms were generated. Both PtPDK transcript abundance and enzyme activity were reduced significantly due to antisense knockdown of PtPDK. Neutral lipid content of transgenic diatom cells increased up to 82% as determined by Nile red staining, and fatty acid composition was not altered. Transgenic cells showed slightly lower growth rate but similar cell size with the wild type, hence retaining similar biomass productivity. CONCLUSIONS: This work first obtained a successful engineered diatom regulating a key gene involved in lipid metabolism. Our findings also provide powerful indications in enhancing microalgal lipid production by metabolic engineering for biofuel industry.

SUBMITTER: Ma YH

PROVIDER: S-EPMC4251698 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Antisense knockdown of pyruvate dehydrogenase kinase promotes the neutral lipid accumulation in the diatom Phaeodactylum tricornutum.

Ma Yu-Han YH Wang Xiang X Niu Ying-Fang YF Yang Zhi-Kai ZK Zhang Meng-Han MH Wang Zhong-Ming ZM Yang Wei-Dong WD Liu Jie-Sheng JS Li Hong-Ye HY

Microbial cell factories 20140809 1

<h4>Background</h4>Microalgae have been an emerging biofuel resource; however, the germplasm improvement has been slow due to the lack of molecular tools. Pyruvate dehydrogenase kinase (PDK) deactivates the pyruvate dehydrogenase complex (PDC) which catalyzes the oxidative decarboxylation of pyruvate. Acetyl-CoA production via PDC is important in plant tissues that are active in fatty acid synthesis.<h4>Results</h4>A 1261-bp cDNA of a putative PDK gene (PtPDK) was cloned from a diatom Phaeodacty ...[more]

PMID: 25106441

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Project description:BACKGROUND:Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30?Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination. RESULTS:The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases). CONCLUSIONS:The results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.

Project description:BackgroundDiatoms, which can accumulate large amounts of carotenoids, are a major group of microalgae and the dominant primary producer in marine environments. Phaeodactylum tricornutum, a model diatom species, acquires little silicon for its growth although silicon is known to contribute to gene regulation and play an important role in diatom intracellular metabolism. In this study, we explored the effects of artificial high-silicate medium (i.e. 3.0 mM sodium metasilicate) and LED illumination conditions on the growth rate and pigment accumulation in P. tricornutum, which is the only known species so far that can grow without silicate. It's well known that light-emitting diodes (LEDs) as novel illuminants are emerging to be superior monochromatic light sources for algal cultivation with defined and efficient red and blue lights.ResultsFirstly, we cultivated P. tricornutum in a synthetic medium supplemented with either 0.3 mM or 3.0 mM silicate. The morphology and size of diatom cells were examined: the proportion of the oval and triradiate cells decreased while the fusiform cells increased with more silicate addition in high-silicate medium; the average length of fusiform cells also slightly changed from 14.33 µm in 0.3 mM silicate medium to 12.20 µm in 3.0 mM silicate medium. Then we cultivated P. tricornutum under various intensities of red light in combination with the two different levels of silicate in the medium. Higher biomass productivity also achieved in 3.0 mM silicate medium than in 0.3 mM silicate medium under red LED light irradiation at 128 μmol/m2/s or higher light intensity. Increasing silicate reversed the down-regulation of fucoxanthin and chlorophyll a under high red-light illumination (i.e. 255 μmol/m2/s). When doubling the light intensity, fucoxanthin content decreased under red light but increased under combined red and blue (50:50) lights while chlorophyll a content reduced under both conditions. Fucoxanthin accumulation and biomass productivity increased with enhanced red and blue (50:50) lights.ConclusionHigh-silicate medium and blue light increased biomass and fucoxanthin production in P. tricornutum under high light conditions and this strategy may be beneficial for large-scale production of fucoxanthin in diatoms.

Dataset Information

Antisense knockdown of pyruvate dehydrogenase kinase promotes the neutral lipid accumulation in the diatom Phaeodactylum tricornutum.

Publications

Antisense knockdown of pyruvate dehydrogenase kinase promotes the neutral lipid accumulation in the diatom Phaeodactylum tricornutum.

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