Bioremoval of Co(II) by a novel halotolerant microalgae Dunaliella sp. FACHB-558 from saltwater.

Cobalt pollution is harmful to both the aquatic ecosystem and human health. As the primary producer of aquatic ecosystems in hypersaline environments, unicellular planktonic Dunaliella microalgae is considered to be a low-energy and eco-friendly biosorbent that removes excess cobalt and enhances the vitality of coastal and marine ecosystems. In this study, we found that the halotolerant microalga named Dunaliella sp. FACHB-558 could grow under a salinity condition with 0.5-4.5 M NaCl. A phylogenetic analysis based on the rbcL gene revealed that Dunaliella sp. FACHB-558 is a close relative of Dunaliella primolecta TS-3. At lab-scale culture, Dunaliella sp. FACHB-558 exhibited high tolerance to heavy metal stresses, including cobalt, nickel, and cadmium. Treatment with 60 µM cobalt delayed its stationary phase but ultimately led to a higher population density. Furthermore, Dunaliella sp. FACHB-558 has the ability to adsorb the cobalt ions in the aquatic environment, which was evidenced by the decreased amount of cobalt in the culture medium. In addition, the tolerance of Dunaliella sp. FACHB-558 to cobalt stress was correlated with enhanced nitric oxide content and peroxidase activity. The autophagy inhibitor 3-MA enhanced nitric oxide burst, increased peroxidase activity, and accelerated the bioremoval of cobalt, suggesting that the autophagy pathway played a negative role in response to cobalt stress in Dunaliella sp. FACHB-558. In summary, our study identified a novel microalga possessing high cobalt tolerance and provided a promising natural biosorbent for the research and application of heavy metal bioremediation technology.

A chromosome-level genome assembly for the astaxanthin-producing microalga Haematococcus pluvialis.

The green microalga Haematococcus pluvialis can synthesize high amounts of astaxanthin, which is a valuable antioxidant that has been utilized in human health, cosmetics, and aquaculture. To illustrate detailed molecular clues to astaxanthin yield, we performed PacBio HIFI along with Hi-C sequencing to construct an improved chromosome-level haplotypic genome assembly with 32 chromosomes and a genome size of 316.0 Mb. Its scaffold N50 (942.6 kb) and contig N50 (304.8 kb) have been upgraded remarkably from our previous genome draft, and a total of 32,416 protein-coding genes were predicted. We also established a high-evidence phylogenetic tree from seven representative algae species, with the main aim to calculate their divergence times and identify expanded/contracted gene families. We also characterized genome-wide localizations on chromosomes of some important genes such as five BKTs (encoding beta-carotene ketolases) that are putatively involved in astaxanthin production. In summary, we reported the first chromosome-scale map of H. pluvialis, which provides a valuable genetic resource for in-depth biomedical investigations on this momentous green alga and commercial astaxanthin bioproduction.

Enhancement of ß-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene ß-cyclase.

ß-Carotene is one of the economically important carotenoids, having functions as the antioxidant to remove harmful free radicals and as the precursor for vitamin A and other high-valued xanthophyll such as zeaxanthin and astaxanthin. Lycopene cyclase plays an important role in the branching of ß-carotene and α-carotene. Aiming to develop the microalgae with enhanced ß-carotene productivity, the CrtY gene from bacterium Pantoea agglomerans was integrated into Chlamydomonas reinhardtii. The lycopene-producing E. coli harboring CrtY gene produced 1.59 times of ß-carotene than that harboring DsLcyb1 from Dunaliella salina (a microalga with abundant ß-carotene), confirming the superior activity of CrtY on ß-carotene biosynthesis. According to the pigment analysis by HPLC, in microalgal transformants that were confirmed by molecular analysis, the expression of CrtY significantly increased ß-carotene content from 12.48 mg/g to 30.65 mg/g (dry weight), which is about 2.45-fold changes. It is noted that three out of five transformants have statistically significant higher amount of lutein, even though the increment was 20% in maximum. Besides, no growth defect was observed in the transformants. This is the first report of functional expression of prokaryotic gene in eukaryotic microalgae, which will widen the gene pool targeting carotenoids biosynthesis using microalgae as the factory and thereby provide more opportunity for high-valued products engineering in microalgae.

Overexpression of 18S rRNA methyltransferase CrBUD23 enhances biomass and lutein content in Chlamydomonas reinhardtii.

Post-transcriptional modification of nucleic acids including transfer RNA (tRNA), ribosomal RNA (rRNA) and messenger RNA (mRNA) is vital for fine-tunning of mRNA translation. Methylation is one of the most widespread post-transcriptional modifications in both eukaryotes and prokaryotes. HsWBSCR22 and ScBUD23 encodes a 18S rRNA methyltransferase that positively regulates cell growth by mediating ribosome maturation in human and yeast, respectively. However, presence and function of 18S rRNA methyltransferase in green algae are still elusive. Here, through bioinformatic analysis, we identified CrBUD23 as the human WBSCR22 homolog in genome of the green algae model organism Chlamydonomas reinhardtii. CrBUD23 was a conserved putative 18S rRNA methyltransferase widely exited in algae, plants, insects and mammalians. Transcription of CrBUD23 was upregulated by high light and down-regulated by low light, indicating its role in photosynthesis and energy metabolism. To characterize its biological function, coding sequence of CrBUD23 fused with a green fluorescence protein (GFP) tag was derived by 35S promoter and stably integrated into Chlamydomonas genome by glass bead-mediated transformation. Compared to C. reinhardtii wild type CC-5325, transgenic strains overexpressing CrBUD23 resulted in accelerated cell growth, thereby leading to elevated biomass, dry weight and protein content. Moreover, overexpression of CrBUD23 increased content of photosynthetic pigments but not elicit the activation of antioxidative enzymes, suggesting CrBUD23 favors growth and proliferation in the trade-off with stress responses. Bioinformatic analysis revealed the G1177 was the putative methylation site in 18S rRNA of C. reinhardtii CC-849. G1177 was conserved in other Chlamydonomas isolates, indicating the conserved methyltransferase activity of BUD23 proteins. In addition, CrTrm122, the homolog of BUD23 interactor Trm112, was found involved in responses to high light as same as CrBUD23. Taken together, our study revealed that cell growth, protein content and lutein accumulation of Chlamydomonas were positively regulated by the 18S rRNA methyltransferase CrBUD23, which could serve as a promising candidate for microalgae genetic engineering.

Research Progress in Heterologous Crocin Production.

Crocin is one of the most valuable components of the Chinese medicinal plant Crocus sativus and is widely used in the food, cosmetics, and pharmaceutical industries. Traditional planting of C. sativus is unable to fulfill the increasing demand for crocin in the global market, however, such that researchers have turned their attention to the heterologous production of crocin in a variety of hosts. At present, there are reports of successful heterologous production of crocin in Escherichia coli, Saccharomyces cerevisiae, microalgae, and plants that do not naturally produce crocin. Of these, the microalga Dunaliella salina, which produces high levels of ß-carotene, the substrate for crocin biosynthesis, is worthy of attention. This article describes the biosynthesis of crocin, compares the features of each heterologous host, and clarifies the requirements for efficient production of crocin in microalgae.

Structure and Phylogeny of Chloroplast and Mitochondrial Genomes of a Chlorophycean Algae Pectinodesmus pectinatus (Scenedesmaceae, Sphaeropleales).

Pectinodesmus pectinatus is a green alga of commercial interest in sewage purification. Clarification of its organelle genomes is helpful for genetic manipulation, taxonomic revisions and evolutionary research. Here, de novo sequencing was used to determine chloroplast genome and mitochondrial genome of P. pectinatus strain F34. The chloroplast genome was composed of a large single-copy (LSC) region of 99,156 bp, a small single-copy (SSC) region of 70,665 bp, and a pair of inverted repeats (IRs) with a length of 13,494 bp each separated by LSC and SSC. The chloroplast genome contained 69 protein-coding genes, 25 transfer-RNA (tRNA) genes, 3 ribosomal RNA (rRNA) genes. The mitochondrial genome was 32,195 bp in length and consisted of 46 unique genes, including 16 protein-coding genes, 27 tRNA genes and 3 rRNA genes. The predominant mutations in organelle genomes were T/A to G/C transitions. Phylogenic analysis indicated P. pectinatus was a sister species to Tetradesmus obliquus and Hariotina sp. within the Pectinodesmus genus. In analysis with CGView Comparison Tool, P. pectinatus organelle genomes displayed the highest sequence similarity with that of T. obliquus. These findings advanced research on the taxonomy and phylogeny of Chlorophyceae algae and particularly revealed the role of P. pectinatus in microalgae evolution.

Molecular cloning and expression analysis of mytilin-like antimicrobial peptides from Asian green mussel Perna viridis.

Mytilin is one of the most important CS-αß peptides involved in innate immune response in Mytilidae. In this study, we successfully identified four mytilin-like antimicrobial peptides (pernalins) from Asian green mussel Perna viridis by aligning the P. viridis transcriptome with 186 mytilins and myticins related sequences collected from the transcriptome data of six Mytilus species. Analysis on gene structure showed that pernalin genes had high conservation with mytilin B of Mediterranean mussel Mytilus galloprovincialis. Interestingly, all pernalin genes have a similar tissue expression feature, evidenced by the highest transcription level observed in the hemocytes and followed by the mantle. The lowest transcription level was observed in the foot and gills. qRT-PCR analysis showed that all pernalin genes were significantly down-regulated at each time points from 3 h to 48 h after Vibrio parahaemolyticus infection, suggesting their timely immune responses after bacterial infection.

Recent advancement and strategy on bio-hydrogen production from photosynthetic microalgae.

Recently, ensuring energy security is a key challenge to political and economic strength in the world. Bio-hydrogen production from microalgae is the promising alternative source for potential renewable and self-sustainability energy but still in the initial phase of development. Practically and sustainability of microalgae hydrogen production is still debatable. The genetic engineering and metabolic pathway engineering of hydrogenase and nitrogenase play a key role to enhance hydrogen production. Microalgae have photosynthetic efficiency and synthesize huge carbohydrate biomass, used as 4th generation feedstock to generate bio-hydrogen. Recent genetically modified strains of microalgae are the attractive source for enhancing bio-hydrogen production in the future. The potential of hydrogen production from microRNAs are gaining great interest of researcher. The main objective of this review is attentive discussed recent approaches on new molecular genetics engineering and metabolic pathway developments, modern photo-bioreactors efficiency, economic assessment, limitations and knowledge gap of bio-hydrogen production from microalgae.

Understanding the functions of endogenous DOF transcript factor in Chlamydomonas reinhardtii.

BACKGROUND: The regulation of genes related to lipid metabolism by genetic engineering is an important way to increase the accumulation of lipids in microalgae. DNA binding with one finger (DOF) is a plant-specific transcription factor in higher plants, where it regulates carbon and nitrogen metabolic pathways by regulating key genes involved in these pathways. Overexpression of DOF can increase lipid production in plants; however, it is not clear whether overexpression of DOF can increase lipids in microalgae. RESULTS: In this study, we cloned a DOF transcription factor, crDOF, from Chlamydomonas reinhardtii. The sequence of this transcription factor is 1875 bp and encodes a peptide of 624 amino acids with a conserved DOF domain. Overexpression of crDOF in C. reinhardtii significantly increased the intracellular lipid content. The content of total fatty acids in the transgenic algae line Tranc-crDOF-12 was 126.01 µg/mg (dry weight), which was 23.24% higher than that of the wild type. Additionally, the content of unsaturated fatty acids in the transgenic Tranc-crDOF-12 line increased significantly. Fluorescence quantitative PCR analysis showed that in the transgenic line Tranc-crDOF-12, the expression levels of BCC1, FAT1, SQD1, MGD1, DGD1 and PGP1 genes were significantly upregulated, while the expression levels of ACP1, ACS1, CIS1 and SQD2 were downregulated. CONCLUSIONS: Our results confirm that crDOF increases intracellular lipids in C. reinhardtii by regulating key genes involved in lipid metabolism. According to these findings, we propose that enhancing the lipid content in microalgae by overexpressing DOF may be achieved in other industrial strains of microalgae and be employed for the industrial production of biodiesel.

Mechanisms of microRNA-mediated gene regulation in unicellular model alga Chlamydomonas reinhardtii.

MicroRNAs are a class of endogenous non-coding RNAs that play a vital role in post-transcriptional gene regulation in eukaryotic cells. In plants and animals, miRNAs are implicated in diverse roles ranging from immunity against viral infections, developmental pathways, molecular pathology of cancer and regulation of protein expression. However, the role of miRNAs in the unicellular model green alga Chlamydomonas reinhardtii remains unclear. The mode of action of miRNA-induced gene silencing in C. reinhardtii is very similar to that of higher eukaryotes, in terms of the activation of the RNA-induced silencing complex and mRNA targeting. Certain studies indicate that destabilization of mRNAs and mRNA turnover could be the major possible functions of miRNAs in eukaryotic algae. Here, we summarize recent findings that have advanced our understanding of miRNA regulatory mechanisms in C. reinhardtii.

The complete chloroplast genome of the green algae Hariotina reticulata (Scenedesmaceae, Sphaeropleales, Chlorophyta).

In this study, the chloroplast genome of Hariotina reticulata was fully sequenced and compared to other Sphaeropleales chloroplast genomes. It is 210,757 bp larger than most Sphaeropleales cpDNAs. It presents a traditional chloroplast structure, and contains 103 genes, including 68 protein-coding genes, six rRNA genes and 29 tRNA genes. The coding region constitutes of 43% of the whole cpDNA. Eighteen introns are found in 11 genes and six introns are unique for Hariotina. 11 open reading frames are identified among these introns. The synteny between Hariotina and Acutodesmus cpDNAs is in general identical, while within Sphaeropleales order, high variability in cpDNA architecture is indicated by general high DCJ distances. Ankyra judayi exhibits the greatest dissimilarity in gene synteny to the others and share some unique gene clusters with Treubaria triappendiculata. The phylogenomic analyses show that A. judayi is clustered with Treubariaceae species and sister to Chlorophyceae incertae sedis and other Sphaeropleales species. The monophyly of Sphaeropleales is rejected.

The far-upstream regulatory region of RFL is required for its precise spatial-temporal expression for floral development in rice.

KEY MESSAGE: A rice mutant aberrant floral organ 1 (afo1) was identified, showing increased floral organ number, aberrant floral organ identity and loss of floral meristem determinacy. A disruption of sequence integrity at 6292-bp upstream of RFL by a T-DNA insertion led to varied RFL expression patterns in floral meristem and floret in afo1 and caused the mutant phenotype. The LEAFY (LFY) transcription factor and its homologs affect many aspects of plant development, especially floral development. RICE FLORICAULA/LEAFY (RFL), the rice ortholog of LFY, has complicated expression patterns and different functions in floral development. However, the mechanisms regulating the spatial-temporal expression of RFL remain largely unknown. Here, we describe a rice aberrant floral organ 1 (afo1) mutant that was produced by a T-DNA insertion at 6292-bp upstream of the start codon of RFL. This insertion altered the expression of RFL in floral meristem (FM) and floret. The in situ hybridization result showed that, when florets appear, RFL was expressed almost exclusively at the palea/lemma adaxial base adjacent to lodicules in the wild-type panicle. However, in afo1 florets, RFL mRNA signals were detected in the region between lodicule and stamen, and strong signals persisted in FM. The altered pattern of RFL expression in afo1 resulted in enlarged FMs, more floral organs, aberrant floral organ identity, and loss of FM determinacy. Transformation of rice with an RFL construct driven by the 6292-bp upstream genomic sequence re-built the mutant phenotype similar to afo1. The results suggest that the far-upstream region of RFL may contain potential cis element(s) that are critical to define the precise spatial-temporal expression pattern of RFL for its function in floral development.

The complete mitochondrial DNA sequence of the green algae Hariotina sp. F30 (Scenedesmaceae, Sphaeropleales, Chlorophyceae).

The complete mitochondrial genome of the green algae Hariotina sp. F30 was obtained in this study using Illumina sequencing data. It is 51 915 bp in length with 36.23% GC content. The genome contains 13 protein-coding genes, 23 tRNA genes and six rRNA genes, all of which are encoded on the heavy strand. AUG is a universal initiation codon among 13 protein-coding genes. UCA is a universal termination codon for most protein-coding gens except UAA in cox1 and cob genes and UGA in nad6 gene. CUU anticodon for tRNA-Lys was detected for the first time in Sphaeropleales.

Complete mitochondrial genome of a DHA-rich protist Schizochytrium sp. TIO1101.

Schizochytrium sp. TIO1101 is a crucial commercial alga used to produce docosahexaenoic acid (DHA), a long-chain polyunsaturated fatty acid that is beneficial for human health. In this study, we sequenced the mitochondrial genome (mitogenome) of Schizochytrium sp. TIO1101 for the first time using an Illumina HiSeq 2500 system (Illumina Inc., San Deigo, CA). The assembled mitogenome was 31 494 bp long with 33.92% GC content. The mitogenome contains 56 genes, including 33 protein-coding genes, 21 transfer RNA genes and two ribosomal RNA genes. Maximum-likelihood phylogenetic analysis of Schizochytrium sp. TIO1101 showed that it was most closely related to Thraustochytrium aureum among the examined species.

An efficient rice mutagenesis system based on suspension-cultured cells.

Plant mutants are important bio-resources for crop breeding and gene functional studies. Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency. Here, we developed a highly-efficient ethyl methanesulfonate (EMS) mutagenesis system based on suspension-cultured cells, with rice (Oryza sativa L.) as an example. We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22 h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations, including a number of important agronomic traits such as grain size, panicle size, grain or panicle shape, tiller number and angle, heading date, male sterility, and disease sensitivity. No mosaic mutant was observed in the mutant lines tested. In this mutant library, we obtained a mutant with an abnormally elongated uppermost internode. Sequencing and functional analysis revealed that this is a new allelic mutant of eui (elongated uppermost internode) caused by two point mutations in the first exon of the EUI gene, representing a successful example of this mutagenesis system.