Youping Xie

PII protein is widely acknowledged to regulate intracellular nitrogen and carbon metabolism by interacting with several crucial proteins. N‐acetyl‐L‐glutamate kinase (NAGK), a rate‐limiting enzyme for arginine biosynthesis, is regarded as a potential target of PII protein. Nevertheless, the regulatory function remains ambiguous in green algae and has not been investigated in Haematococcus pluvialis . In this study, the NAGK enzyme and PII protein of H. pluvialis (designated as HpNAGK and HpPII, respectively) and their interaction relationships were characterized. The results indicated that HpNAGK showed high similarity with the same enzyme in the green algae. A subcellular localization assay indicated that both HpPII and HpNAGK were located in the chloroplasts. Yeast two‐hybrid, pull‐down, and bimolecular fluorescence complementation assays distinctly verified the interaction between HpPII and HpNAGK, which occurs in the chloroplasts. The structure of the HpPII‐HpNAGK complex was predicted through docking analysis. Moreover, the HpNAGK activity was significantly enhanced by HpPII in the presence of glutamine in vitro. Under nitrogen starvation, HpNAGK activity declined in vivo, concomitant with a reduction in arginine accumulation. The regulatory function of HpPII on HpNAGK activity aligned with that in Chlamydomonas reinhardtii but differed from that in Dunaliella salina , suggesting species specificity among green algae. These findings provide insights into the regulatory function of PII protein in green algae and help to unveil the response mechanisms of H. pluvialis to different nitrogen statuses.

Lutein is a high-value carotenoid with many human health benefits. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of lycopene into distinct downstream branches, one of which is the lutein biosynthesis pathway, via α-carotene. Hence, LCYB and LCYE are key enzymes in lutein biosynthesis. In this study, the coding genes of two lycopene cyclases (CsLCYB and CsLCYE) of a lutein-enriched marine green microalga, Chlorella sorokiniana FZU60, were isolated and identified. A sequence analysis and computational modeling of CsLCYB and CsLCYE were performed using bioinformatics to identify the key structural domains. Further, a phylogenetic analysis revealed that CsLCYB and CsLCYE were homogeneous to the proteins of other green microalgae. Subcellular localization tests in Nicotiana benthamiana showed that CsLCYB and CsLCYE localized in chloroplasts. A pigment complementation assay in Escherichia coli revealed that CsLCYB could efficiently β-cyclize both ends of lycopene to produce β-carotene. On the other hand, CsLCYE possessed a strong ε-monocyclase activity for the production of δ-carotene and a weak ε-bicyclic activity for the production of ε-carotene. In addition, CsLCYE was able to catalyze lycopene into β-monocyclic γ-carotene and ultimately produced α-carotene with a β-ring and an ε-ring via γ-carotene or δ-carotene. Moreover, the co-expression of CsLCYB and CsLCYE in E. coli revealed that α-carotene was a major product, which might lead to the production of a high level of lutein in C. sorokiniana FZU60. The findings provide a theoretical foundation for performing metabolic engineering to improve lutein biosynthesis and accumulation in C. sorokiniana FZU60.

Background: Chlorella sorokinianaFZU60 is a promising lutein producing microalga. A mixotrophy/photoautotrophy two-stage strategy can achieve high biomass concentration at stage 1 and high lutein content at stage 2, leading to excellent lutein production efficiency inC. sorokinianaFZU60. However, the underlying molecular mechanisms are still unclear, restraining the further improvement of lutein production.Results: In this study, physiological and biochemical analysis revealed that photochemical parameters (Fv/Fm and NPQ) and photosynthetic pigments contents increased during the shift from mixotrophy to photoautotrophy, indicating that photosynthesis and photoprotection enhanced. Furthermore, transcriptomic analysis revealed that the glyoxylate cycle and TCA cycle were suppressed after the shift to photoautotrophy, leading to a decreased cell growth rate. However, the gene expression levels of photosynthesis, CO₂fixation, autophagy, and lutein biosynthesis were upregulated at the photoautotrophy stage, demonstrating that microalgal cells could obtain more precursor to synthesize lutein for enhancing photosynthesis and reducing reactive oxygen species.Conclusions: The findings help to elucidate the molecular mechanisms for high lutein production efficiency ofC. sorokinianaFZU60 under the mixotrophy/photoautotrophy strategy, identify key functional genes responsible for lutein biosynthesis, and shed light on further improvement of lutein production by genetic or metabolic engineering in future studies.

Co-production of multiple compounds is an efficient approach to enhance the economic feasibility of microalgae-based metabolites production. In this study, Chlorella sorokiniana FZU60 was cultivated under different bioprocess strategies to enhance the co-production of lutein and protein. Results showed that both lutein and protein content (7.72 and 538.06 mg/g, respectively) were highest at the onset of nitrogen deficiency under batch cultivation. Semi-batch III strategy, with 75% microalgal culture replacement by fresh medium, obtained similar content, productivity, and yield of lutein and protein as batch cultivation, demonstrating that it can be used for stable and continuous production. Fed-batch II strategy, feeding with 1/3 modified BG11 medium, achieved super-high lutein and protein yield (28.81 and 1592.77 mg/L, respectively), thus can be used for high-output production. Besides, two-stage strategy, combining light intensity shift and semi-batch cultivation, gained extremely high lutein and protein productivity (15.31 and 1080.41 mg/L/day, respectively), thereby is a good option for high-efficiency production. Moreover, the fed-batch II and two-stage strategy achieved high-quality lutein and protein, thus are promising for the co-production of lutein and protein in C. sorokiniana FZU60 for commercial application.

Marine microalgae are regarded as potential feedstock because of their multiple valuable compounds, including lipids, pigments, carbohydrates, and proteins. Some of these compounds exhibit attractive bioactivities, such as carotenoids, ω-3 polyunsaturated fatty acids, polysaccharides, and peptides. However, the production cost of bioactive compounds is quite high, due to the low contents in marine microalgae. Comprehensive utilization of marine microalgae for multiple compounds production instead of the sole product can be an efficient way to increase the economic feasibility of bioactive compounds production and improve the production efficiency. This paper discusses the metabolic network of marine microalgal compounds, and indicates their interaction in biosynthesis pathways. Furthermore, potential applications of co-production of multiple compounds under various cultivation conditions by shifting metabolic flux are discussed, and cultivation strategies based on environmental and/or nutrient conditions are proposed to improve the co-production. Moreover, biorefinery techniques for the integral use of microalgal biomass are summarized. These techniques include the co-extraction of multiple bioactive compounds from marine microalgae by conventional methods, super/subcritical fluids, and ionic liquids, as well as direct utilization and biochemical or thermochemical conversion of microalgal residues. Overall, this review sheds light on the potential of the comprehensive utilization of marine microalgae for improving bioeconomy in practical industrial application.

Microalgae are considered as excellent candidates for bioactive compounds, yet microalgal residues remaining after the extraction of one or two compounds are usually discarded, which is not economical. This study demonstrates the alkaline extraction of proteins from Chlorella pyrenoidosa residue after lipid and pigment extractions, and their functional properties. Single-factor experiments and response surface methodology were used to obtain the optimal conditions for protein extraction. Based on our results, a maximum protein yield of 722.70 mg/g, was obtained under the following extraction conditions: sodium hydroxide concentration 7.90%, extraction temperature 70.00 °C, extraction time 34.80 min, and microalgal residue concentration 8.20 mg/mL. The molecular weight of microalgal residue protein isolate (MRPI) was mainly distributed at the regions of 0.18–0.50 kDa, 0.50–1.50 kDa, and 1.50–5.00 kDa. The essential amino acid content was greater than the values recommended by FAO/WHO standards; a high essential amino acid index value (1.49) was another good indication that MRPI is suitable for human consumption. Moreover, MRPI exhibited excellent emulsifying properties and antioxidant activity, which suggests it may be useful as an emulsifying agent and antioxidant. These findings could improve the extraction methods of functional protein from microalgal residue and add value to microalgae-based bioactive compound production processes.

Marine microalgae has great potential for lutein production with the advantage of saving fresh water resource. Thus, marine microalga Chlamydomonas sp. JSC4 is investigated as a potential lutein producer in this study. The medium types, nitrate‐N and sea salt concentration are individually investigated to promote the cell growth rate and lutein production of JSC4. In Modified Bold Basal 3N medium, cell growth and lutein content are optimal at the nitrate‐N concentration of 1000 mg L⁻¹ and sea salt concentration of 2%. In addition, an innovative salinity‐gradient strategy is operated to dramatically enhance biomass productivity (560 mg/L/d) and lutein content (3.42 mg g⁻¹), resulting in the optimal lutein productivity (1.92 mg/L/d). Overall, this study clearly demonstrates that salinity is a significant inducer of lutein accumulation by strain JSC4 and that lutein production can be successfully optimized using the salinity‐gradient strategy, which is beneficial for the outdoor large‐scale lutein production in the future.