Synthetic biology in microalgae towards fucoxanthin production for pharmacy and nutraceuticals.

Synthetic biology has emerged as a powerful tool for engineering biological systems to produce valuable compounds, including pharmaceuticals and nutraceuticals. Microalgae, in particular, offer a promising platform for the production of bioactive compounds due to their high productivity, low land and water requirements, and ability to perform photosynthesis. Fucoxanthin, a carotenoid pigment found predominantly in brown seaweeds and certain microalgae, has gained significant attention in recent years due to its numerous health benefits, such as antioxidation, antitumor effect and precaution osteoporosis. This review provides an overview of the principles and applications of synthetic biology in the microbial engineering of microalgae for enhanced fucoxanthin production. Firstly, the fucoxanthin bioavailability and metabolism in vivo was introduced for the beneficial roles, followed by the biological functions of anti-oxidant activity, anti-inflammatory activity, antiapoptotic role antidiabetic and antilipemic effects. Secondly, the cultivation condition and strategy were summarized for fucoxanthin improvement with low production costs. Thirdly, the genetic engineering of microalgae, including gene overexpression, knockdown and knockout strategies were discussed for further improving the fucoxanthin production. Then, synthetic biology tools of CRISPR-Cas9 genome editing, transcription activator-like effector nucleases as well as modular assembly and chassis engineering were proposed to precise modification of microalgal genomes to improve fucoxanthin production. Finally, challenges and future perspectives were discussed to realize the industrial production and development of functional foods of fucoxanthin from microalgae.

Integrated metabolic tools reveal carbon alternative in Isochrysis zhangjiangensis for fucoxanthin improvement.

This study explored the regulation of photosystem and central carbon metabolism in cell growth and fucoxanthin accumulation of Isochrysis zhangjiangensis via transcriptome analysis, targeted metabolite measurements, and flux balance analysis. High light promoted biomass accumulation but dramatically decreased fucoxanthin productivity. It suppressed the active photosystem and reduced chlorophyll content, but improved metabolic flux of Calvin-Benson-Bassham and tricarboxylic acid cycle for massive biomass accumulation. The CO2 fixation was largely dependent on mitochondrial energy illustrated by the integrated metabolic tools. At a molecular level, glyceraldehyde-3-phosphate, acetyl-CoA, and pyruvate contents increased at exponential phase under high light, which tended to participate into fatty acid biosynthesis by the up-regulated ACCase. However, high light inhibited most genes involved in fucoxanthin biosynthesis and induced diadinoxanthin cycle to diatoxanthin form. Therefore, constant light at 100 µmol m-2 s-1 balancing biomass concentration and fucoxanthin content provided the highest fucoxanthin productivity at 3.06 mg L-1 d-1.

Coordinating Carbon Metabolism and Cell Cycle of Chlamydomonasreinhardtii with Light Strategies under Nitrogen Recovery.

Nutrient supplementation is common in microalgae cultivation to enhance the accumulation of biomass and biofunctional products, while the recovery mechanism from nutrient starvation is less investigated. In this study, the influence of remodeled carbon metabolism on cell cycle progression was explored by using different light wavelengths under N-repletion and N-recovery. The results suggested that blue light enhanced cell enlargement and red light promoted cell division under N-repletion. On the contrary, blue light promoted cell division by stimulating cell cycle progression under N-recovery. This interesting phenomenon was ascribed to different carbon metabolisms under N-repletion and N-recovery. Blue light promoted the recovery of photosystem II and redirected carbon skeletons into proteins under N-recovery, which potentially accelerated cell recovery and cell cycle progression. Although red light also facilitated the recovery of photosystem II, it mitigated the degradation of polysaccharide and then arrested almost all the cells in the G1 phase. By converting light wavelengths at the 12 h of N-recovery with blue light, red and white lights were proved to increase biomass concentration better than continuous blue light. These results revealed different mechanisms of cell metabolism of Chlamydomonas reinhardtii during N-recovery and could be applied to enhance cell vitality of microalgae from nutrient starvation and boost biomass production.

Functional Characterization of Long-Chain Acyl-CoA Synthetase Gene Family from the Oleaginous Alga Chromochloris zofingiensis.

Long-chain acyl-coenzyme A (CoA) synthetase (LACS) catalyzes the formation of acyl-CoAs from free fatty acids, which is pivotal for lipid metabolism. Here, we confirmed the presence of six CzLACS genes in Chromochloris zofingiensis. Functional complementation and in vitro enzymatic assay indicated that CzLACS2 through CzLACS5 rather than CzLACS1 or CzLACS6 are bona fide LACS enzymes and they have overlapping yet distinct substrate preference. The results of the subcellular colocalization experiment and different expression patterns under three triacylglycerol (TAG)-inducing conditions showed that CzLACS2 through CzLACS4 reside at endoplasmic reticulum (ER) and are involved in TAG biosynthesis, while CzLACS5 resides in peroxisome and participates in fatty acid ß-oxidation. The yeast one-hybrid assay using a library of 50 transcription factors (TFs) constructed in our study identified 12 TFs potentially involved in regulating the expression of CzLACSs. Moreover, heterologous expression of CzLACSs demonstrated their engineering potential for modulating TAG synthesis in yeast and algal cells.

Fucoxanthinol from the Diatom Nitzschia Laevis Ameliorates Neuroinflammatory Responses in Lipopolysaccharide-Stimulated BV-2 Microglia.

In recent years, microalgae have drawn increasing attention as a valuable source of functional food ingredients. Intriguingly, Nitzschia laevis is rich in fucoxanthinol that is seldom found in natural sources. Fucoxanthinol, a marine xanthophyll carotenoid, possesses various beneficial bioactivities. Nevertheless, it's not clear whether fucoxanthinol could exert anti-neuroinflammatory function. In light of these premises, the aim of the present study was to investigate the anti-inflammatory role of fucoxanthinol purified from Nitzschia laevis in Lipopolysaccharide (LPS)-stimulated microglia. The results showed that pre-treatment of fucoxanthinol remarkably attenuated the expression of LPS-induced nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the production of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE-2), nitric oxide (NO) and reactive oxygen species (ROS) induction. Modulation mechanism studies revealed that fucoxanthinol hampered nuclear factor-kappa B (NF-κB), Akt, and mitogen-activated protein kinase (MAPK) pathways. Meanwhile, fucoxanthinol led to the enhancement of nuclear translocation of NF-E2-related factor 2 (Nrf2), and the upregulation of heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO-1). Taken together, the results indicated that fucoxanthinol obtained from Nitzschia laevis had great potential as a neuroprotective agent in neuroinflammation and neurodegenerative disorders.

Characterization of Microalgal Acetyl-CoA Synthetases with High Catalytic Efficiency Reveals Their Regulatory Mechanism and Lipid Engineering Potential.

Acetyl-CoA synthetase (ACS) plays a key role in microalgal lipid biosynthesis and acetyl-CoA industrial production. In the present study, two ACSs were cloned and characterized from the oleaginous microalga Chromochloris zofingiensis. In vitro kinetic analysis showed that the Km values of CzACS1 and CzACS2 for potassium acetate were 0.99 and 0.81 mM, respectively. Moreover, CzACS1 and CzACS2 had outstanding catalytic efficiencies (kcat/Km), which were 70.67 and 79.98 s-1 mM-1, respectively, and these values were higher than that of other reported ACSs. CzACS1 and CzACS2 exhibited differential expression patterns at the transcriptional level under various conditions. Screening a recombinant library of 52 transcription factors (TFs) constructed in the present study via yeast one-hybrid assay pointed to seven TFs with potential involvement in the regulation of the two ACS genes. Expression correlation analysis implied that GATA20 was likely an important regulator of CzACS2 and that ERF9 could regulate two CzACSs simultaneously.

Storage carbon metabolism of Isochrysis zhangjiangensis under different light intensities and its application for co-production of fucoxanthin and stearidonic acid.

This study explored the co-production of fucoxanthin and stearidonic acid from Isochrysis zhangjiangensis by investigating its carbon metabolism under different light intensities. Results showed high light inhibited the synthesis of fucoxanthin and stearidonic acid, while promoted cell growth and enhanced cellular lipid content compared with low light, achieving 2.4 g/L and 28.55%, respectively. Low light accelerated the accumulation of fucoxanthin and stearidonic acid, which obtained 23.29 mg/g and 17.16% (of total fatty acid). In combination with the molecular analysis, low light redirected carbon skeletons into glyceraldehyde-3-phosphate and diverted into carotenoid especially fucoxanthin. While, high light redistributed the skeletons to Malonyl CoA, citrate and α-Ketoglutarate and then oriented into lipid metabolism. The highest fucoxanthin and stearidonic acid productivity was 2.94 mg L-1 d-1 and 4.33 mg L-1 d-1, respectively, which revealed I. zhanjiangensis is a potential strain for the co-production of fucoxanthin and stearidonic acid.

A novel strategy for isolation and purification of fucoxanthinol and fucoxanthin from the diatom Nitzschia laevis.

In this study, the microalga Nitzschia laevis (N. laevis) can accumulate a marine carotenoid fucoxanthinol. In particular, fucoxanthinol was firstly isolated from microalgae, accompanied by its derivative fucoxanthin. The identification and quantification of fucoxanthinol and fucoxanthin were determined by ultra-performance liquid chromatography coupled to photodiode array detector-quadrupole/travelling-wave ion mobility mass spectrometry/time-of-flight mass spectrometry (UPLC-PDA-TWIMS-QTOF-MS). Furthermore, a cost-effective approach mediated with solid-phase extraction (SPE) and thin-layer chromatography (TLC) technique was used to isolate and purify fucoxanthinol and fucoxanthin from the extracts of N. laevis. This two-step method can obtain 98% fucoxanthinol and 95% fucoxanthin, with the recovery efficiencies of around 85% for fucoxanthinol and 70% for fucoxanthin, respectively. Moreover, 1H and 13C nuclear magnetic resonance (NMR) techniques were adopted to record the purified compounds for supporting the above results. In all, the developed method has a promising potential to purify fucoxanthinol and fucoxanthin of microalgae for food and pharmaceutical applications.

Correction to: High-value biomass from microalgae production platforms: strategies and progress based on carbon metabolism and energy conversion.

[This corrects the article DOI: 10.1186/s13068-018-1225-6.].

High-value biomass from microalgae production platforms: strategies and progress based on carbon metabolism and energy conversion.

Microalgae are capable of producing sustainable bioproducts and biofuels by using carbon dioxide or other carbon substances in various cultivation modes. It is of great significance to exploit microalgae for the economical viability of biofuels and the revenues from high-value bioproducts. However, the industrial performance of microalgae is still challenged with potential conflict between cost of microalgae cultivation and revenues from them, which is mainly ascribed to the lack of comprehensive understanding of carbon metabolism and energy conversion. In this review, we provide an overview of the recent advances in carbon and energy fluxes of light-dependent reaction, Calvin-Benson-Bassham cycle, tricarboxylic acid cycle, glycolysis pathway and processes of product biosynthesis in microalgae, with focus on the increased photosynthetic and carbon efficiencies. Recent strategies for the enhanced production of bioproducts and biofuels from microalgae are discussed in detail. Approaches to alter microbial physiology by controlling light, nutrient and other environmental conditions have the advantages of increasing biomass concentration and product yield through the efficient carbon conversion. Engineering strategies by regulating carbon partitioning and energy route are capable of improving the efficiencies of photosynthesis and carbon conversion, which consequently realize high-value biomass. The coordination of carbon and energy fluxes is emerging as the potential strategy to increase efficiency of carbon fixation and product biosynthesis. To achieve more desirable high-value products, coordination of multi-stage cultivation with engineering and stress-based strategies occupies significant positions in a long term.

Lutein suppresses inflammatory responses through Nrf2 activation and NF-κB inactivation in lipopolysaccharide-stimulated BV-2 microglia.

SCOPE: In this study, the effects of lutein on neuroinflammation in lipopolysaccharide (LPS)-activated BV-2 microglia were investigated. METHODS AND RESULTS: The production of proinflammatory cytokines tumor necrosis factor-α, interleukin-1ß, and nitric oxide was measured in culture medium using enzyme immunoassay and Griess reagent, respectively. The expression of proteins was determined using Western blot. Pretreatment with lutein (50 µM) prior to LPS (1 µg/mL, 12 h) stimulation resulted in a significant inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression, as well as tumor necrosis factor-α, interleukin-1ß, and nitric oxide production (p < 0.05). Further experiments demonstrated that lutein suppressed LPS-induced NF-κB activation by inhibiting the phosphorylation of p38 kinase, c-Jun N-terminal kinase (JNK), and Akt kinase (p < 0.05). Moreover, lutein markedly quenched reactive oxygen species and promoted antioxidant protein expression including heme oxygenase-1 and NAD(P)H: quinone oxidoreductase by enhancing the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) mediated NF-E2-related factor 2 (Nrf2) activation (p < 0.05). CONCLUSION: These results suggest that lutein attenuates neuroinflammation in LPS-activated BV-2 microglia partly through inhibiting p38-, JNK-, and Akt-stimulated NF-κB activation and promoting ERK-induced Nrf2 activation, suggesting that lutein has great potential as a nutritional preventive strategy in inflammation-related neurodegenerative disorders.