Supplementation with rac-GR24 Facilitates the Accumulation of Biomass and Astaxanthin in Two Successive Stages of Haematococcus pluvialis Cultivation.

The unicellular freshwater green alga Haematococcus pluvialis has attracted much research attention due to its biosynthetic ability for large amounts of astaxanthin, a blood-red ketocarotenoid that is used in cosmetics, nutraceuticals, and pharmaceuticals. Recently, numerous studies have investigated the functions of natural astaxanthin; however, the high cost of the production of astaxanthin from H. pluvialis cultures restricts its commercial viability. There is an urgent need to fulfill commercial demands by increasing astaxanthin accumulation from H. pluvialis cultures. In this study, we discovered that treatment of H. pluvialis cultures at the beginning of the macrozooid stage (day 0) with 1 µM rac-GR24, a synthetic analogue of strigolactones (a class of phytohormones), led to significant increases in biomass [up to a maximum dry cell weight (DCW) of 0.53 g/L] during the macrozooid stage and astaxanthin (from 0.63 to 5.32% of DCW) during the hematocyst stage. We elucidated that this enhancement of biomass accumulation during the macrozooid stage by rac-GR24 is due to its increasing CO2 utilization efficiency in photosynthesis and carbohydrate biosynthesis. We also found that rac-GR24 stimulated the overproduction of nicotinamide adenine dinucleotide phosphate (NADPH) and antioxidant enzymes in H. pluvialis cultures, which alleviated the oxidative damage caused by reactive oxygen species generated during the hematocyst stage due to the exhaustion of nitrogen supplies. Moreover, rac-GR24 treatment of H. pluvialis synergistically altered the activity of the pathways of fatty acid biosynthesis and astaxanthin esterification, which resulted in larger amounts of astaxanthin being generated by rac-GR24-treated cultures than by controls. In summary, we have developed a feasible and economic rac-GR24-assisted strategy that increases the amounts of biomass and astaxanthin generated by H. pluvialis cultures, and have provided novel insights into the mechanistic roles of rac-GR24 to achieve these effects.

Effective bioremediation of tobacco wastewater by microalgae at acidic pH for synergistic biomass and lipid accumulation.

Tobacco wastewater is too difficult to decontaminate which poses a significant environmental problem due to the harmful and toxic components. Chlorella pyrenoidosa is a typical microalgal species with potential in removal of organic/inorganic pollutants and proves to be an ideal algal-based system for wastewater treatment. However, the strategy of tobacco related wastewater treatment using microalgae is in urgent need of development. In this study, C. pyrenoidosa was used to evaluate the removal efficiency of artificial tobacco wastewater. Under various solid-to-liquid (g/L) ratios, 1:1 ratio and acidic pH 5.0 were optimal for C. pyrenoidosa to grow with high performance of removal capacity to toxic pollutants (such as COD, NH3-N, nicotine, nitrosamines and heavy metals) with the alleviation of oxidative damage. Algal biomass could reach up to 540.24 mg/L. Furthermore, carbon flux of C. pyrenoidosa was reallocated from carbohydrate and protein biosynthesis to lipogenesis with a high lipid content of 268.60 mg/L at pH 5.0. Overall, this study demonstrates an efficient and sustainable strategy for tobacco wastewater treatment at acidic pH with the production of valuable microalgal products, which provides a promising biorefinery strategy for microalgal-based wastewater bioremediation.

Hyperaccumulation of fucoxanthin by enhancing methylerythritol phosphate pathway in Phaeodactylum tricornutum.

The established human health benefits of carotenoids along with the contemporary consumption of natural carotenoids bring the necessity to sustainable production of carotenoids. Among, marine diatoms have emerged as the potential biological resources for carotenoid production; however, their relatively lower yield in native strains provides the impetus to genetically improve the diatoms to cope with the burgeoning demand. In this study, we genetically improved the diatom Phaeodactylum tricornutum by overexpressing key carotenogenic genes involved in methylerythritol phosphate (MEP) pathway. The genes with lower relative transcript level under optimum conditions such as CMK and CMS were selected and overexpressed in P. tricornutum individually. Both CMK and CMS overexpressing lines exhibited elevated growth and photosynthesis. The expression of key carotenogenic genes such as PSY, PDS, ZDS, CRT, and LCYB was significantly upregulated. Furthermore, total carotenoid content was significantly increased; particularly, fucoxanthin content was increased by 1.83- and 1.82-fold in engineered lines CMK and CMS, respectively. Together, the results identify the potential metabolic targets and also uncover the crucial role of MEP pathway in redirecting metabolic precursors towards carotenogenesis. KEY POINTS: • Low abundant genes CMS and CMK of MEP pathway were overexpressed in the diatom • Total carotenoid content was increased, particularly fucoxanthin • Critical metabolic nodes were uncovered to accelerate fucoxanthin biosynthesis.

Overproduction of Bioactive Algal Chrysolaminarin by the Critical Carbon Flux Regulator Phosphoglucomutase.

Chrysolaminarin, the primary polysaccharide reservoir in some marine algae, has attracted much attention due to its broad health properties. However, its biosynthetic pathway and regulation mechanisms have rarely been reported which hinders the improvement of production efficiency. Therefore, this study aims to identify key metabolic nodes in the chrysolaminarin biosynthetic pathway. A phosphoglucomutase (PGM) in the model microalga Phaeodactylum tricornutum, revealing its critical role in chrysolaminarin biosynthesis is identified. PGM overexpression significantly elevates chrysolaminarin content by 2.54-fold and reaches 25.6% of cell dry weight; while algal growth and photosynthesis are not impaired. Besides, PGM overexpression up- and down-regulates the expression of chrysolaminarin and lipid biosynthetic genes, respectively. Microscopic analysis of aniline blue stained cells reveals that overproduced chrysolaminarin localized predominantly in vacuoles. Lipidomic analyses reveal that PGM overexpression significantly reduces the lipid content. The findings reveal the critical role of PGM in regulating the carbon flux between carbohydrate and lipid biosynthesis in microalgae, and provide a promising candidate for high efficiency production of chrysolaminarin.

Light-harvesting protein Lhcx3 is essential for high light acclimation of Phaeodactylum tricornutum.

The light-harvesting protein complexes (Lhc) play key roles in the processes of light absorption and protection in diatoms. However, different Lhc protein carries out distinct function in photosynthesis. For now, roles of many Lhc proteins in light acclimation are largely unknown. Here, function of Lhcx3 in marine diatom Phaeodactylum tricornutum was examined by using reverse genetic technologies. The overexpression of Lhcx3 led to increased diadinoxanthin + diatoxanthin content and elevated non-photochemical fluorescence quenching (NPQ) while knockdown of Lhcx3 reduced NPQ level. In addition, the expression of Lhcx3 could be induced by blue light but not by red light. After addition of the photosynthetic inhibitor, upregulation of Lhcx3 transcript in high light could be inhibited by NH4Cl, but not by DCMU (3-(3,4-dichlorophenyl)-l,l-dim ethylurea). In contrast, DCMU addition increased expression of Lhcx3 in high light. In combination with changes of NPQ after addition of inhibitor, we concluded that the Lhcx3 played key roles in high light acclimation of diatoms. This finding will provide new clues for genetic improvement of P. tricornutum with an aim to cultivate new strains with high growth rate.

Constitutive and Chloroplast Targeted Expression of Acetyl-CoA Carboxylase in Oleaginous Microalgae Elevates Fatty Acid Biosynthesis.

Photosynthetic microalgae are of burgeoning interest in the generation of commercial bioproducts. Microalgae accumulate high lipid content under adverse conditions, which in turn compromise their growth and hinder their commercial potential. Hence, it is necessary to engineer microalgae to mitigate elevated lipid accumulation and biomass. In this study, we identified acetyl-CoA carboxylase (ACCase) in oleaginous microalga Phaeodactylum tricornutum (PtACC2) and expressed constitutively in the chloroplast to demonstrate the potential of chloroplast engineering. Molecular characterization of transplastomic microalgae revealed that PtACC2 was integrated, transcribed and expressed successfully, and localized in the chloroplast. Enzymatic activity of ACCase was elevated by 3.3-fold, and the relative neutral lipid content increased substantially by 1.77-fold, and lipid content reached up to 40.8% of dry weight. Accordingly, the number and size of oil bodies markedly increased. Fatty acid profiling showed that the content of monounsaturated fatty acids increased, while polyunsaturated fatty acids decreased. This method provides a valuable genetic engineering toolbox for microalgal bioreactors with industrial significance.

Enrichment of Long-Chain Polyunsaturated Fatty Acids by Coordinated Expression of Multiple Metabolic Nodes in the Oleaginous Microalga Phaeodactylum tricornutum.

Microalgal long-chain polyunsaturated fatty acids (LC-PUFAs) have emerged as promising alternatives to depleting fish oils. However, the overproduction of LC-PUFAs in microalgae has remained challenging. Here, we report a sequential metabolic engineering strategy that systematically overcomes the metabolic bottlenecks and overproduces LC-PUFAs. Malonyl CoA-acyl carrier protein transacylase, catalyzing the first committed step in type II fatty acid synthesis, and desaturase 5b, involved in fatty acid desaturation, were coordinately expressed in Phaeodactylum tricornutum. Engineered microalgae hyper-accumulated LC-PUFAs, with arachidonic acid (ARA) and docosahexaenoic acid (DHA) contents of up to 18.98 µg/mg and 9.15 µg/mg (dry weight), respectively. Importantly, eicosapentaenoic acid (EPA) was accumulated up to a highest record of 85.35 µg/mg by metabolic engineering. ARA and EPA were accumulated mainly in triacylglycerides, whereas DHA was found exclusively in phospholipids. Combinatorial expression of these critical enzymes led to the optimal increment of LC-PUFAs without unbalanced metabolic flux and demonstrated the practical feasibility of generating sustainable LC-PUFA production.