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Anti-lipopolysaccharide factor 3 (ALF Pm 3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALF Pm 3 is limited by its low production in nature, as well as its low activity when expressed in Escherichia coli and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALF Pm 3 in Chlamydomonas reinhardtii . In this study, signal peptides ARS1 and CAH1 were fused with ALF Pm 3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to C. reinhardtii JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALF Pm 3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALF Pm 3 could be detected in algal cells and culture medium by immunoblot, meaning that ALF Pm 3 was successfully expressed in C. reinhardtii and secreted into the extracellular environment. Moreover, ALF Pm 3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of V. harveyi , V. alginolyticus , V. anguillarum , and V. parahaemolyticus within 24 h. Interestingly, the inhibitory rate of c-ALF Pm 3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALF Pm 3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALF Pm 3 peptide. Our results provided a new strategy for the secretory production of ALF Pm 3 with high antibacterial activity in C. reinhardtii , which could improve the application potentiality of ALF Pm 3 in the aquaculture industry.

Microalgae-based biodiesel production has many advantages over crude oil extraction and refinement, thus attracting more and more concern. Protein ubiquitination is a crucial mechanism in eukaryotes to regulate physiological responses and cell development, which is highly related to algal biodiesel production. Cullins as the molecular base of cullin-RING E3 ubiquitin ligases (CRLs), which are the largest known class of ubiquitin ligases, control the life activities of eukaryotic cells. Here, three cullins (CrCULs) in the green microalgae Chlamydomonas reinhardtii were identified and characterized. To investigate the roles of CrCULs in lipid metabolism, the gene expression profiles of CrCUL s under nutrition starvation were examined. Except for down-regulation under nitrogen starvation, the CrCUL3 gene was induced by sulfur and iron starvation. CrCUL2 seemed insensitive to nitrogen and sulfur starvation because it only had changes after treatment for eight days. CrCUL4 exhibited an expression peak after nitrogen starvation for two days but this declined with time. All CrCUL s expressions significantly increased under iron deficiency at two and four days but decreased thereafter. The silencing of CrCUL2 and CrCUL4 expression using RNAi (RNA interference) resulted in biomass decline and lipids increase but an increase of 20% and 28% in lipid content after growth for 10 days, respectively. In CrCUL2 and CrCUL4 RNAi lines, the content of fatty acids, especially C16:0 and C18:0, notably increased as well. However, the lipid content and fatty acids of the CrCUL3 RNAi strain slightly changed. Moreover, the subcellular localization of CrCUL4 showed a nuclear distribution pattern. These results suggest CrCUL2 and CrCUL4 are regulators for lipid accumulation in C. reinhardtii . This study may offer an important complement of lipid biosynthesis in microalgae.

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene ( ALFPm3 ), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii , which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii -derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon , providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.

Anti-lipopolysaccharide factor is a class of antimicrobial peptides with lipopolysaccharide-binding structural domains, which has a broad antimicrobial spectrum, high antimicrobial activities, and broad application prospects in terms of the aquaculture industry. However, the low yield of natural antimicrobial peptides and their poor expression activity in bacteria and yeast have hindered their exploration and utilization. Therefore, in this study, the extracellular expression system of Chlamydomonas reinhardtii, by fusing the target gene with the signal peptide, was used to express anti-lipopolysaccharide factor 3 (ALFPm3) from Penaeus monodon in order to obtain highly active ALFPm3. Transgenic C. reinhardtii T-JiA2, T-JiA3, T-JiA5, and T-JiA6, were verified using DNA-PCR, RT-PCR, and immunoblot. Additionally, the IBP1-ALFPm3 fusion protein could be detected not only within the cells but also in the culture supernatant. Moreover, the extracellular secretion containing ALFPm3 was collected from algal cultures, and then its bacterial inhibitory activity was analyzed. The results showed that the extracts from T-JiA3 had an inhibition rate of 97% against four common aquaculture pathogenic bacteria, including Vibrio harveyi, Vibrio anguillarum, Vibrio alginolyticus, and Vibrio parahaemolyticus. The highest inhibition rate of 116.18% was observed in the test against V. anguillarum. Finally, the minimum inhibition concentration (MIC) of the extracts from T-JiA3 to V. harveyi, V. anguillarum, V. alginolyticus, and V. parahaemolyticus were 0.11 μg/μL, 0.088 μg/μL, 0.11 μg/μL, and 0.011 μg/μL, respectively. This study supports the foundation of the expression of highly active anti-lipopolysaccharide factors using the extracellular expression system in C. reinhardtii, providing new ideas for the expression of highly active antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Microalgae lipid triacylglycerol is considered as a promising feedstock for national production of biofuels. A hotspot issue in the biodiesel study is to increase TAG content and productivity of microalgae. Precursor RNA processing protein (Prp19), which is the core component of eukaryotic RNA splice NTC (nineteen associated complex), plays important roles in the mRNA maturation process in eukaryotic cells, has a variety of functions in cell development, and is even directly involved in the biosynthesis of oil bodies in mouse. Nevertheless, its function in Chlamydomonas reinhardtii remains unknown. Here, transcriptional level of CrPrp19 under nutrition deprivation was analyzed, and both its RNA interference and overexpressed transformants were constructed. The expression level of CrPrp19 was suppressed by nitrogen or sulfur deficiency. Cell densities of CrPrp19 RNAi lines decreased, and their neutral lipid contents increased 1.33 and 1.34 times over those of controls. The cells of CrPrp19 RNAi lines were larger and more resistant to sodium acetate than control. Considerably none of the alterations in growth or neutral lipid contents was found in the CrPrp19 overexpression transformants than wild type. Fatty acids were also significantly increased in CrPrp19 RNAi transformants. Subcellular localization and yeast two-hybrid analysis showed that CrPrp19 was a nuclear protein, which might be involved in cell cycle regulation. In conclusion, CrPrp19 protein was necessary for negatively regulating lipid enrichment and cell size, but not stimulatory for lipid storage. [ABSTRACT FROM AUTHOR]

Many microalgae could accumulate high level of astaxanthin, especially a green microalga Haematococcus pluvialis . β-carotene ketolase gene ( bkt ) is a key enzyme for astaxanthin biosynthesis in Haematococcus pluvialis , and astaxanthin accumulation could be induced effectively by abiotic stresses such as high concentration of sodium acetate and high light. In Haematococcus there are 3 bkt gene copies, among them, the transcription level of bkt1 was obviously correlated with high light and sodium acetate. Thus, the function of bkt1 promoter played a key role in understanding the regulatory mechanisms of astaxanthin biosynthesis. A TATA-box and cis -acting elements associated with high light and stress-related responses were detected in bkt1 promoter in previously studies. However, detailed information about cis -acting regulatory elements of this important promoter is still unavailable since a reliable genetic manipulation technique is not available yet. To identify the functions of cis -elements in bkt1 promoter, 8 constructs contained different length of bkt1 promoter were successfully introduced into a model microalga Chlamydomonas reinhardtii and different transformants were obtained with zeomycin resistant selection. Exposed to high light or sodium acetate, differentially increased ble transcripts and protein production in transformants indicated that these cis -elements respond to high light and sodium acetate. Moreover, we found that TATA-box in bkt1 promoter is essential for the promoter function and ble expression was correlated with cis -acting elements in bkt1 promoter. Our results shed lights on regulation mechanisms of the key genes from Haematococcus for astaxanthin biosynthesis in Chlamydomonas reinhardtii . [ABSTRACT FROM AUTHOR]

A cell-wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC-849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris-acetate-phosphate media containing 10 μg · mL−1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly-3-hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol · min−1 · mg protein−1 to 126 nmol · min−1 · mg protein−1. The amount of PHB in double transgenic algae was determined by gas chromatography–mass spectrometry (GC–MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation. [ABSTRACT FROM AUTHOR]

The papain-like cysteine proteases (PLCPs), the most important group of cysteine proteases, have been reported to participate in the regulation of growth, senescence, and abiotic stresses in plants. However, the functions of PLCPs and their roles in stress response in microalgae was rarely reported. The responses to different abiotic stresses in Haematococcus pluvialis were often observed, including growth regulation and astaxanthin accumulation. In this study, the cDNA of HpXBCP3 containing 1515 bp open reading frame (ORF) was firstly cloned from H. pluvialis by RT-PCR. The analysis of protein domains and molecular evolution showed that HpXBCP3 was closely related to AtXBCP3 from Arabidopsis. The expression pattern analysis revealed that it significantly responds to NaCl stress in H. pluvialis. Subsequently, transformants expressing HpXBCP3 in Chlamydomonas reinhardtii were obtained and subjected to transcriptomic analysis. Results showed that HpXBCP3 might affect the cell cycle regulation and DNA replication in transgenic Chlamydomonas, resulting in abnormal growth of transformants. Moreover, the expression of HpXBCP3 might increase the sensitivity to NaCl stress by regulating ubiquitin and the expression of WD40 proteins in microalgae. Furthermore, the expression of HpXBCP3 might improve chlorophyll content by up-regulating the expression of NADH-dependent glutamate synthases in C. reinhardtii. This study indicated for the first time that HpXBCP3 was involved in the regulation of cell growth, salt stress response, and chlorophyll synthesis in microalgae. Results in this study might enrich the understanding of PLCPs in microalgae and provide a novel perspective for studying the mechanism of environmental stress responses in H. pluvialis. [ABSTRACT FROM AUTHOR]

The expression vector containing phbB and ble genes was constructed and transformed into cell-wall-deficient strain Chlamydomonas reinhardtii CC-849 by the glass-bead method. The transgenic alga was selected and maintained in the TAP agar plates containing 10 μg/mL Zeomycin. Transgenic alga, which could express phbB at the transcriptional level, was obtained and further confirmed with PCR, Southern blot and RT-PCR-DNA hybridization analysis. [ABSTRACT FROM AUTHOR]

Background: Sulfur-deprived cultivation of Chlamydomonas reinhardtii, referred as “two-stage culture” transferring the cells from regular algal medium to sulfur-deplete one, has been extensively studied to improve photobio-H2 production in this green microalga. During sulfur-deprivation treatment, the synthesis of a key component of photosystem II complex, D1 protein, was inhibited and improved photobio-H2 production could be established in C. reinhardtii. However, separation of algal cells from a regular liquid culture medium to a sulfur-deprived one is not only a discontinuous process, but also a cost- and time-consuming operation. More applicable and economic alternatives for sustained H2 production by C. reinhardtii are still highly required. Results: In the present study, a significant improvement in photobio-H2 production was observed in the transgenic green microalga C. reinhardtii, which employed a newly designed strategy based on a heat-inducible artificial miRNA (amiRNA) expression system targeting D1-encoded gene, psbA. A transgenic algal strain referred as “amiRNA-D1” has been successfully obtained by transforming the expression vector containing a heat-inducible promoter. After heat shock conducted in the same algal cultures, the expression of amiRNA-D1 was detected increased 15-fold accompanied with a 73% decrease of target gene psbA. More interestingly, this transgenic alga accumulated about 60% more H2 content than the wild-type strain CC-849 at the end of 7-day cultivation. Conclusions: The photobio-H2 production in the engineered transgenic alga was significantly improved. Without imposing any nutrient-deprived stress, this novel strategy provided a convenient and efficient way for regulation of photobio-H2 production in green microalga by simply “turn on” the expression of a designed amiRNA. [ABSTRACT FROM AUTHOR]