Widespread Sterol Methyltransferase Participates in the Biosynthesis of Both C4α- and C4ß-Methyl Sterols.

The 4-methyl steranes serve as molecular fossils and are used for studying both eukaryotic evolution and geological history. The occurrence of 4α-methyl steranes in sediments has long been considered evidence of products of partial demethylation mediated by sterol methyl oxidases (SMOs), while 4ß-methyl steranes are attributed entirely to diagenetic generation from 4α-methyl steroids since possible biological sources of their precursor 4ß-methyl sterols are unknown. Here, we report a previously unknown C4-methyl sterol biosynthetic pathway involving a sterol methyltransferase rather than the SMOs. We show that both C4α- and C4ß-methyl sterols are end products of the sterol biosynthetic pathway in an endosymbiont of reef corals, Breviolum minutum, while this mechanism exists not only in dinoflagellates but also in eukaryotes from alveolates, haptophytes, and aschelminthes. Our discovery provides a previously untapped route for the generation of C4-methyl steranes and overturns the paradigm that all 4ß-methyl steranes are diagenetically generated from the 4α isomers. This may facilitate the interpretation of molecular fossils and understanding of the evolution of eukaryotic life in general.

Improved growth of bait microalgae Isochrysis and aquacultural wastewater treatment with mixotrophic culture.

This research of mixotrophic microalgae Isochrysis 3011 with glycerol was combined with the treatment of aqua-cultural wastewater, different initial concentrations, and optimized light intensities. The algae growth rate, removal efficiencies of total nitrogen (TN) and total phosphorus (TP) were determined. Results showed that the suitable initial concentration was 0.4 g L-1, and the optimum light intensity was 60 µmol m-2 s-1. The growth of the mixotrophic group was better than that of the autotrophic culture. The biomass yield of the mixotrophic group with glycerol was 0.17 g L-1 d-1, and the removal rates of TN and TP were 73.39% and 95.61%, respectively. The content of total lipid and total protein in mixotrophic group were higher than the values of the autotrophic group. This indicates that aquaculture wastewater treatment with mixotrophic bait microalgae can obtain superior micro-algal biomass, which is also a potential technology for wastewater utilization and ecological protection.

Manipulating fatty-acid profile at unit chain-length resolution in the model industrial oleaginous microalgae Nannochloropsis.

The chain length (CL) of fatty acids (FAs) is pivotal to oil property, yet to what extent it can be customized in industrial oleaginous microalgae is unknown. In Nannochloropsis oceanica, to modulate long-chain FAs (LCFAs), we first discovered a fungi/bacteria-originated polyketide synthase (PKS) system which involves a cytoplasmic acyl-ACP thioesterase (NoTE1). NoTE1 hydrolyzes C16:0-, C16:1- and C18:1-ACP in vitro and thus intercepts the specific acyl-ACPs elongated by PKS for polyunsaturated FA biosynthesis, resulting in elevation of C16/C18 monounsaturated FAs when overproduced and increase of C20 when knocked out. For medium-chain FAs (MCFAs; C8-C14), C8:0 and C10:0 FAs are boosted by introducing a Cuphea palustris acyl-ACP TE (CpTE), whereas C12:0 elevated by rationally engineering CpTE enzyme's substrate-binding pocket to shift its CL preference towards C12:0. A mechanistic model exploiting both native and engineered PKS and type II FAS pathways was thus proposed for manipulation of carbon distribution among FAs of various CL. The ability to tailor FA profile at the unit CL resolution from C8 to C20 in Nannochloropsis spp. lays the foundation for scalable production of designer lipids via industrial oleaginous microalgae.

Engineering microalgae: transition from empirical design to programmable cells.

Domesticated microalgae hold great promise for the sustainable provision of various bioresources for human domestic and industrial consumption. Efforts to exploit their potential are far from being fully realized due to limitations in the know-how of microalgal engineering. The associated technologies are not as well developed as those for heterotrophic microbes, cyanobacteria, and plants. However, recent studies on microalgal metabolic engineering, genome editing, and synthetic biology have immensely helped to enhance transformation efficiencies and are bringing new insights into this field. Therefore, this article, summarizes recent developments in microalgal biotechnology and examines the prospects for generating specialty and commodity products through the processes of metabolic engineering and synthetic biology. After a brief examination of empirical engineering methods and vector design, this article focuses on quantitative transformation cassette design, elaborates on target editing methods and emerging digital design of algal cellular metabolism to arrive at high yields of valuable products. These advances have enabled a transition of manners in microalgal engineering from single-gene and enzyme-based metabolic engineering to systems-level precision engineering, from cells created with genetically modified (GM) tags to that without GM tags, and ultimately from proof of concept to tangible industrial applications. Finally, future trends are proposed in microalgal engineering, aiming to establish individualized transformation systems in newly identified species for strain-specific specialty and commodity products, while developing sophisticated universal toolkits in model algal species.

Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9.

Microalgae are promising feedstock for biofuels yet mechanistic probing of their cellular network and industrial strain development have been hindered by lack of genome-editing tools. Nannochloropsis spp. are emerging model microalgae for scalable oil production and carbon sequestration. Here we established a CRISPR/Cas9-based precise genome-editing approach for the industrial oleaginous microalga Nannochloropsis oceanica, using nitrate reductase (NR; g7988) as example. A new screening procedure that compares between restriction enzyme-digested nested PCR (nPCR) products derived from enzyme-digested and not-digested genomic DNA of transformant pools was developed to quickly, yet reliably, detect genome-engineered mutants. Deep sequencing of nPCR products directly amplified from pooled genomic DNA revealed over an 1% proportion of 5-bp deletion mutants and a lower frequency of 12-bp deletion mutants, with both types of editing precisely located at the targeted site. The isolated mutants, in which precise deletion of five bases caused a frameshift in NR translation, grow normally under NH4 Cl but fail to grow under NaNO3 , and thus represent a valuable chassis strain for transgenic-strain development. This demonstration of CRISPR/Cas9-based genome editing in industrial microalgae opens many doors for microalgae-based biotechnological applications.

Antagonistic roles of abscisic acid and cytokinin during response to nitrogen depletion in oleaginous microalga Nannochloropsis oceanica expand the evolutionary breadth of phytohormone function.

The origin of phytohormones is poorly understood, and their physiological roles in microalgae remain elusive. Genome comparison of photosynthetic autotrophic eukaryotes has revealed that the biosynthetic pathways of abscisic acid (ABA) and cytokinins (CKs) emerged in unicellular algae. While ABA and CK degradation mechanisms emerged broadly in algal lineages, complete vascular plant-type conjugation pathways emerged prior to the rise of Streptophyta. In microalgae, a complete set of proteins from the canonical ABA and CK sensing and signaling pathways is not essential, but individual components are present, suggesting stepwise recruitment of phytohormone signaling components. In the oleaginous eustigmatophyte Nannochloropsis oceanica IMET1, UHPLC-MS/MS detected a wide array of plant hormones, despite a phytohormone profile that is very distinct from that of flowering plants. Time-series transcriptional analysis during nitrogen depletion revealed activation of the ABA biosynthetic pathway and antagonistic transcription of CK biosynthetic genes. Correspondingly, the ABA level increases while the dominant bioactive CK forms decrease. Moreover, exogenous CKs stimulate cell-cycle progression while exogenous ABA acts as both an algal growth repressor and a positive regulator in response to stresses. The presence of such functional flowering plant-like phytohormone signaling systems in Nannochloropsis sp. suggests a much earlier origin of phytohormone biosynthesis and degradation than previously believed, and supports the presence in microalgae of as yet unknown conjugation and sensing/signaling systems that may be exploited for microalgal feedstock development.

Towards Lipid from Microalgae: Products, Biosynthesis, and Genetic Engineering.

Microalgae can convert carbon dioxide into organic matter through photosynthesis. Thus, they are considered as an environment-friendly and efficient cell chassis for biologically active metabolites. Microalgal lipids are a class of organic compounds that can be used as raw materials for food, feed, cosmetics, healthcare products, bioenergy, etc., with tremendous potential for commercialization. In this review, we summarized the commercial lipid products from eukaryotic microalgae, and updated the mechanisms of lipid synthesis in microalgae. Moreover, we reviewed the enhancement of lipids, triglycerides, polyunsaturated fatty acids, pigments, and terpenes in microalgae via environmental induction and/or metabolic engineering in the past five years. Collectively, we provided a comprehensive overview of the products, biosynthesis, induced strategies and genetic engineering in microalgal lipids. Meanwhile, the outlook has been presented for the development of microalgal lipids industries, emphasizing the significance of the accurate analysis of lipid bioactivity, as well as the high-throughput screening of microalgae with specific lipids.

The Performance Analysis of Grouting Repair Effect on the Accuracy of Disturbance Stress Test in Damaged Surrounding Rock Mass.

Disturbance stress assessment is crucial for ensuring the safety of deep engineering projects. Currently, the primary technique for continuously monitoring three-dimensional disturbance stress is the stress relief method, but its accuracy can be compromised by rock damage that occurs after excavation. To mitigate this issue, grouting is employed to repair damaged rock masses and enhance their mechanical properties. However, the impact of grouting techniques on improving the accuracy of disturbance stress testing is challenging to evaluate through laboratory and in situ experiments. To address this problem, numerical simulation technology is employed to investigate disturbance stress testing after the repair of damaged surrounding rock through grouting. The simulation results indicate that grouting repair significantly enhances the accuracy of stress testing. As the depth of damaged rock mass repair increases, the error in stress testing decreases. Achieving complete repair of the initial damage zone during grouting is essential to eliminate errors in stress testing. Expanding on the positive effects of grouting repair on stress testing, a segmented testing method for disturbance stress is proposed. The method involves separately testing the initial stress and stress changes, thereby reducing the stress level within the rock, minimizing rock failure, and enhancing the accuracy of disturbance stress testing. This study provides valuable reference methods, and the outcomes of this research will serve as a foundation for enhancing the accuracy of disturbance stress testing in deep hard rock engineering.

Solving the Jigsaw puzzle of phytosterol diversity by a novel sterol methyltransferase from Zea mays.

Phytosterols are vital structural and regulatory components in plants. Zea mays produces a series of phytosterols that are specific to corn. However, the underline biosynthetic mechanism remains elusive. In this study, we identified a novel sterol methyltransferase from Z. mays (ZmSMT1-2) which showed a unique feature compared with documented plant SMTs. ZmSMT1-2 showed a substrate preference for cycloartenol. Using S-adenosyl-L-methionine (AdoMet) as a donor, ZmSMT1-2 converted cycloartenol into alkylated sterols with unique side-chain architectures, including Δ25(27) (i.e., cyclolaudenol and cycloneolitsol) and Δ24(25) (i.e., cyclobranol) sterols. Cycloneolitsol is identified as a product of SMTs for the first time. Our discovery provides a previously untapped mechanism for phytosterol biosynthesis and adds another layer of diversity of sterol biosynthesis.

Synergy between microalgae and microbiome in polluted waters.

Microalga-microbiome interactions are central to both health and disease of aquatic environments. Despite impressive advances in deciphering how microorganisms participate in and impact aquatic ecosystems, the evolution and ecological involvement of microalgae and the microbiome in polluted waters are typically studied independently. Here, the phycosphere (i.e., the consortia of microalgae and the related microbiome) is regarded as an independent and integrated life form, and we summarize the survival strategies exhibited by this symbiont when exposed to anthropogenic pollution. We highlight the cellular strategies and discuss the modulation at the transcriptional and population levels, which reciprocally alters community structure or genome composition for medium-term acclimation or long-term adaptation. We propose a 'PollutantBiome' concept to help the understanding of microalga-microbiome interactions and development of beneficial microbial synthetic communities for pollutant remediation.

Control Phytophagous Nematodes By Engineering Phytosterol Dealkylation Caenorhabditis elegans as a Model.

Plant-parasitic nematodes ingest and convert host phytosterols via dealkylation to cholesterol for both structural and hormonal requirements. The insect 24-dehydrocholesterol reductase (DHCR24) was shown in vitro as a committed enzyme in the dealkylation via chemical blocking. However, an increased brood size and ovulation rate, instead compromised development, were observed in the engineered nematode Caenorhabditis elegans where the DHCR24 gene was knocked down, indicating the relationship between DHCR24 and dealkylation and their function in nematodes remains illusive. In this study, a defect in C. elegans DHCR24 causes impaired growth of the nematode with sitosterol (a major component of phytosterols) as a sole sterol source. Plant sterols with rationally designed structure (null substrates for dealkylation) can't be converted to cholesterol in wild-type worms, and their development was completely halted. This study underpins the essential function of DHCR24 in nematodes and would be beneficial for the development of novel nematocidal strategies.

Engineering a marine microalga Chlorella sp. as the cell factory.

The use of marine microalgae in industrial systems is attractive for converting CO2 into value-added products using saline water and sunlight. The plant nature and demonstrated industrial potential facilitate Chlorella spp. as excellent model organisms for both basic research and commercial application. However, the transformation method has not been developed in marine Chlorella spp., thus genetic engineering is hindered in exploiting the industrial potentialities of these strains. In this study, we provided a transformation protocol for the marine Chlorella strain MEM25, which showed robust characteristics, including high production of proteins and polyunsaturated fatty acids in multiple cultivation systems over various spatial-temporal scales. We showed that transformants could be obtained in a dramatically time-saving manner (comparable to Saccharomyces cerevisiae) with four functional proteins expressed properly. The transgenes are integrated into the genome and can be successfully inherited for more than two years. The development of a marine Chlorella transformation method, in combination with the complete genome, will greatly facilitate more comprehensive mechanism studies and provide possibilities to use this species as chassis for synthetic biology to produce value-added compounds with mutual advantage in neutralization of CO2 in commercial scales.

Artificial switches induce the bespoke production of functional compounds in marine microalgae Chlorella by neutralizing CO2.

To improve the CO2 tolerance of a marine microalga Chlorella sp. of which the production capacity has been demonstrated industrially, a mutant library was created and a strain hct53 was screened. Compared to the parental strain, hct53 shows a high CO2 capture capacity, while starch biosynthesis is compromised, with increases in health beneficial metabolites and antioxidant capacity. Global gene expression and genome-wide mutation distribution revealed that transcript choreography was concomitant with more active CO2 sequestration, an increase in the lipid synthesis, and a decrease in the starch and protein synthesis. These results suggest that artificial trait improvement via mutagenesis, couple with multiomics analysis, helps discover genetic switches that induce the bespoke conversion of carbon flow from "redundant metabolites" to valuable ones for functional food.

A Functional Genomics View of Gibberellin Metabolism in the Cnidarian Symbiont Breviolum minutum.

Dinoflagellate inhabitants of the reef-building corals exchange nutrients and signals with host cells, which often benefit the growth of both partners. Phytohormones serve as central hubs for signal integration between symbiotic microbes and their hosts, allowing appropriate modulation of plant growth and defense in response to various stresses. However, the presence and function of phytohormones in photosynthetic dinoflagellates and their function in the holobionts remain elusive. We hypothesized that endosymbiotic dinoflagellates may produce and employ phytohormones for stress responses. Using the endosymbiont of reef corals Breviolum minutum as model, this study aims to exam whether the alga employ analogous signaling systems by an integrated multiomics approach. We show that key gibberellin (GA) biosynthetic genes are widely present in the genomes of the selected dinoflagellate algae. The non-13-hydroxylation pathway is the predominant route for GA biosynthesis and the multifunctional GA dioxygenase in B. minutum has distinct substrate preference from high plants. GA biosynthesis is modulated by the investigated bleaching-stimulating stresses at both transcriptional and metabolic levels and the exogenously applied GAs improve the thermal tolerance of the dinoflagellate. Our results demonstrate the innate ability of a selected Symbiodiniaceae to produce the important phytohormone and the active involvement of GAs in the coordination and the integration of the stress response.

Sustainable development of microalgal biotechnology in coastal zone for aquaculture and food.

Region-specific Research and Development (R&D) of microalga-derived product systems are crucial if "biotech's green gold" is to be explored in a rational and economically viable way. Coastal zones, particularly the locations around the equator, are typically considered to be optimum cultivation sites due to stable annual temperature, light, and ready availability of seawater. However, a 'cradle-to-grave' assessment of the development of microalgal biotechnology in these areas, not only under the laboratory conditions, but also in the fields has not yet been demonstrated. In this study, to evaluate the viability of microalga-derived multi-product technology, we showed the development of microalgal biotechnology in coastal zones for aquaculture and food. By creating and screening a (sub)tropical microalgal collection, a Chlorella strain MEM25 with a robust growth in a wide range of salinities, temperatures, and light intensities was identified. Evaluation of the economic viability and performance of different scale cultivation system designs (500 L and 5000 L closed photobioreactors and 60,000 L open race ponds, ORPs) at coastal zones under geographically specific conditions showed the stable and robust characteristics of MEM25 across different production system designs and various spatial and temporal scales. It produces high amounts of proteins and polyunsaturated fatty acids (PUFAs) in various conditions. Feeding experiments reveal the nutritional merits of MEM25 as food additives where PUFAs and essential amino acids are enriched and the algal diet improves consumers' growth. Economic evaluation highlights an appreciable profitability of MEM25 production as human or animal food using ORP systems. Therefore, despite the pros and cons, sound opportunities exist for the development of market-ready multiple-product systems by employing region-specific R&D strategies for microalgal biotechnology.

Role of an ancient light-harvesting protein of PSI in light absorption and photoprotection.

Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli.

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

Improving lipid productivity by engineering a control-knob gene in the oleaginous microalga Nannochloropsis oceanica.

Nannochloropsis spp. are promising industrial microalgae for scalable oil production and the lipid production can be boosted by nutrient starvation and high irradiance. However, these stimuli halt growth, thereby decreasing overall productivity. In this study, we created transgenic N. oceanica where AtDXS gene encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS) derived from Arabidopsis thaliana was overexpressed in vivo. Compared with the wild type (WT), engineered Nannochloropsis showed a higher CO2 absorption capacity and produced more biomass, lipids, and carbohydrates with more robust growth in either preferred conditions or various stressed conditions (low light, high light, nitrogen starvation, and trace element depletion). Specifically, relative to the WT, lipid production increased by ~68.6% in nitrogen depletion (~1.08 â€‹g â€‹L-1) and ~110.6% in high light (~1.15 â€‹g â€‹L-1) in the transgenic strains. As for neutral lipid (triacylglycerol, TAG), the engineered strains produced ~93.2% more in nitrogen depletion (~0.77 â€‹g â€‹L-1) and ~148.6% more in high light (~0.80 â€‹g â€‹L-1) than the WT. These values exceed available records in engineered industrial microalgae. Therefore, engineering control-knob genes could modify multiple biological processes simultaneously and enable efficient carbon partitioning to lipid biosynthesis with elevated biomass productivity. It could be further exploited for simultaneous enhancement of growth property and oil productivity in more industrial microalgae.

Clade-Specific Sterol Metabolites in Dinoflagellate Endosymbionts Are Associated with Coral Bleaching in Response to Environmental Cues.

Cnidarians cannot synthesize sterols (which play essential roles in growth and development) de novo but often use sterols acquired from endosymbiotic dinoflagellates. While sterol availability can impact the mutualistic interaction between coral host and algal symbiont, the biosynthetic pathways (in the dinoflagellate endosymbionts) and functional roles of sterols in these symbioses are poorly understood. In this study, we found that itraconazole, which perturbs sterol metabolism by inhibiting the sterol 14-demethylase CYP51 in dinoflagellates, induces bleaching of the anemone Heteractis crispa and that bleaching perturbs sterol metabolism of the dinoflagellate. While Symbiodiniaceae have clade-specific sterol metabolites, they share features of the common sterol biosynthetic pathway but with distinct architecture and substrate specificity features of participating enzymes. Tracking sterol profiles and transcripts of enzymes involved in sterol biosynthesis across time in response to different environmental cues revealed similarities and idiosyncratic features of sterol synthesis in the endosymbiont Breviolum minutum Exposure of algal cultures to high levels of light, heat, and acidification led to alterations in sterol synthesis, including blocks through downregulation of squalene synthase transcript levels accompanied by marked growth reductions.IMPORTANCE These results indicate that sterol metabolites in Symbiodiniaceae are clade specific, that their biosynthetic pathways share architectural and substrate specificity features with those of animals and plants, and that environmental stress-specific perturbation of sterol biosynthesis in dinoflagellates can impair a key mutualistic partnership for healthy reefs.

Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7.

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15 degrees C and 3.9-fold at 4 degrees C compared to the alga at 25 degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.