The fate of antibiotic resistance genes in wastewater containing microalgae treated by chlorination, ultra-violet, and Fenton reaction.

Antibiotic resistance genes (ARGs) and bacteria (ARBs) in the effluent of wastewater treatment plants (WWTPs) are of utmost importance for the dissemination of ARGs in natural aquatic environments. Therefore, there is an urgent need for effective technologies to eliminate WWTP ARGs/ARBs and mitigate the associated risks posed by the discharged ARG in aquatic environments. To test the effective technology for eliminating ARGs/ARBs, we compared the removal of ARGs and ARBs by three different tertiary treatments, namely ultra-violet (UV) disinfection, chlorination disinfection, and Fenton oxidation. Then, the treated wastewater was co-cultured with Chlorella vulgaris (representative of aquatic biota) to investigate the fate of discharged ARGs into the aquatic environment. The results demonstrated that chlorination (at a chlorine concentration of 15 mg/L) and Fenton (at pH 2.73, with 0.005 mol/L Fe2+ and 0.0025 mol/L H2O2) treatment showed higher efficacy in ARG removal (1.8 - 4.17 logs) than UV treatment (15 min) (1.29 - 3.87 logs). Moreover, chlorine at 15 mg/L and Fenton treatment effectively suppressed ARB regeneration while UV treatment for 15 min could not. Regardless of treatments tested in this study, the input of treated wastewater to the Chlorella system increased the number of ARGs and mobile genetic elements (MGEs), indicating the potential risk of ARG dissemination associated with WWTP discharge. Among the wastewater-Chlorella co-culture systems, chlorination resulted in less of an increase in the number of ARGs and MGEs compared to Fenton and UV treatment. When comparing the wastewater systems to the co-culture systems, it was observed that Chlorella vulgaris reduced the number of ARGs and MGEs in chlorination and UV-treated wastewater; however, Chlorella vulgaris promoted ARG survival in Fenton-treated water, suggesting that aquatic microalgae might act as a barrier to ARG dissemination. Overall, chlorination treatment not only effectively removes ARGs and inhibits ARB regeneration but also shows a lower risk of ARG dissemination. Therefore, chlorination is recommended for practical application in controlling the spread of discharged ARGs from WWTP effluent in natural aquatic environments.

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris.

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Efficacy of White Spot Syndrome Virus Protein VP28-Expressing Chlorella vulgaris as an Oral Vaccine for Shrimp.

The white spot syndrome virus (WSSV) is the causative agent of white spot disease, which kills shrimp within a few days of infection. Although WSSV has a mortality rate of almost 100% and poses a serious threat to the shrimp farming industry, strategies for its prevention and treatment are extremely limited. In this study, we examined the efficacy of VP28, a recombinant WSSV protein expressed in Chlorella vulgaris (C. vulgaris), as an oral shrimp vaccine. When compared with the control group, in which WSSV had a cumulative mortality of 100%, shrimp treated with 5% VP28-expressing C. vulgaris in their feed only had a 20% cumulative mortality rate 12 days after the WSSV challenge. When compared with the nonvaccinated group, the transcription of anti-lipopolysaccharide factor, C-type lectin, and prophenoloxidase genes, which are involved in shrimp defense against WSSV infection, was upregulated 29.6 fold, 15.4 fold, and 11.5 fold, respectively. These findings highlight C. vulgaris as a potential host for industrial shrimp vaccine production.

Fatty acid based antimicrobials from Streptomyces sp. SORS-24, an endophyte isolated from Sonchus oleraceus.

Due to the rise in bacterial resistance towards various therapeutic agents, interest is now developing towards fatty acid based antimicrobials because of their non-specific mode of action. A strain SORS 24 isolated from Sonchus oleraceus (Sow thistle) showed significant activity against Escherichia coli ATCC 25922 (25 mm), Chlorella vulgaris (20 mm), Bacillus subtilis DSM 10 (ATCC 6051) and Pseudomonas sp. (15 mm). It displayed an LC50 value of 10 µg/ml against Artemia salina (Brine shrimp) nauplii and an EC50 value of 0.8 µg/ml in the (DPPH) diphenylpicrylhydrazyl antioxidant assay. The strain also displayed genotoxicity against a PolA deficient strain, E. coli K-12 AB 3027 (15 mm). Mass spectrometry (HPLC-MS) showed that the strain produced oleamide (9-Octadecenamide) and erucamide (13-Docosenamide). Both of the purified fatty acid amides showed prominent activity against B. subtilis DSM 10 (ATCC 6051) (20 mm) and E. coli ATCC 25922 (15 mm). Significant genotoxicity was observed against E. coli K-12 AB 3027 (15 mm). The 16S gene sequencing revealed that the strain belonged to species, Streptomyces tanashiensis. As far as our understanding, this is the first report of this species producing these fatty acid based antimicrobials.

Isolation and Characterization of a Salt Inducible Promoter from Chlorella vulgaris PKVL7422.

Chlorella is a eukaryotic organism that can be used as an industrial host to produce recombinant proteins. In this study, a salt-inducible promoter (SIP) was isolated from the freshwater species Chlorella vulgaris PKVL7422 from the screening of genes that were upregulated after salt treatment. Several cis-acting elements, including stress response elements, were identified in the isolated SIP. Moreover, the Gaussia luciferase gene was cloned after the SIP and transformed into C. vulgaris to test the inducibility of this promoter. Reexamination of transcriptome of C. vulgaris revealed that genes involved in the synthesis of methyl jasmonic acid (MeJA), gibberellin (GA), and abscisic acid (ABA) were upregulated when C. vulgaris was treated with salt. Furthermore, the expression level of recombinant luciferase increased when the transformed C. vulgaris was treated with salt and MeJA, GA, and ABA. This study represents the first report of the C. vulgaris SIP and highlights how transformed microalgae could be used for robust expression of recombinant proteins.

Optimization of nutrient utilization efficiency and productivity for algal cultures under light and dark cycles using genome-scale model process control.

Algal cultivations are strongly influenced by light and dark cycles. In this study, genome-scale metabolic models were applied to optimize nutrient supply during alternating light and dark cycles of Chlorella vulgaris. This approach lowered the glucose requirement by 75% and nitrate requirement by 23%, respectively, while maintaining high final biomass densities that were more than 80% of glucose-fed heterotrophic culture. Furthermore, by strictly controlling glucose feeding during the alternating cycles based on model-input, yields of biomass, lutein, and fatty acids per gram of glucose were more than threefold higher with cycling compared to heterotrophic cultivation. Next, the model was incorporated into open-loop and closed-loop control systems and compared with traditional fed-batch systems. Closed-loop systems which incorporated a feed-optimizing algorithm increased biomass yield on glucose more than twofold compared to standard fed-batch cultures for cycling cultures. Finally, the performance was compared to conventional proportional-integral-derivative (PID) controllers. Both simulation and experimental results exhibited superior performance for genome-scale model process control (GMPC) compared to traditional PID systems, reducing the overall measured value and setpoint error by 80% over 8 h. Overall, this approach provides researchers with the capability to enhance nutrient utilization and productivity of cell factories systematically by combining genome-scale models and controllers into an integrated platform with superior performance to conventional fed-batch and PID methodologies.

Algal-bacterial consortium mediated system offers effective removal of nitrogen nutrients and antibiotic resistance genes.

The sulfonamide antibiotic resistance genes (ARGs) especially sul1 was identified as the dominant in eutrophic water. The performance of Chlorella vulgaris-B. licheniformis consortium toward sul1 removal, total nitrogen (TN) removal, and the mechanism of sul1 removal was investigated. The removal efficiency of exogenous ARGs plasmids carrying sul1 reached (97.2 ± 2.3)%. The TN removal rate reached (98.5 ± 1.2)%. The enhancements of carbon metabolism, nitrogen metabolism, aminoacyl-tRNA biosynthesis, and glycoproteins had significant influences on sul1 and TN removals, under the premise of normal growth of algae and bacteria. The quantitative polymerase chain reaction (qPCR) results suggested that the absolute abundances of sul1 were low in algal-bacterial systems (0 gene copies/mL) compared with individual systems ((1 × 106 ± 15) gene copies/mL). The duplication of sul1 was inhibited in algal cells and bacterial cells. The algal-bacterial consortium seems to be a promising technology for wastewater treatment with a potential to overcome the eutrophication and ARGs challenges.

Weighted gene Co-expression network analysis (WGCNA) reveals a set of hub genes related to chlorophyll metabolism process in chlorella (Chlorella vulgaris) response androstenedione.

Androstenedione (ADSD) was the main androgen detected in wastewaters. Chlorella was the most widely used plant in biological wastewater treatment process. In order to understand the toxicological response of chlorella to ADSD contamination, we used the weighted gene co-expression network analysis (WGCNA) method to systematically analyze the gene regulatory networks of chlorella after ADSD treatments. Total of 25 modules was identified from gene co-expression networks, and the turquoise module were selected for GO and KEGG enrichment analysis. Results showed that most hub genes were associated with chloroplast organizations or photosystems processes. Among them, the expressions profiles of hcar, nol, pao and sgr genes were highly correlated to the content fluctuations of chlorophylls after different ADSD treatments. All these results demonstrated that chlorophylls play a key role in preventing cell damage of chlorella caused by ADSD contamination. Besides, we proposed a possible chlorophyll metabolism pathway in chlorella response to ADSD contamination.

Transcriptome analysis of potential flocculation-related genes in Streptomyces sp. hsn06 with flocculation activity on Chlorella vulgaris biomass.

Chlorella vulgaris is a biomass energy provider with promising potential to help alleviate the energy crisis. Streptomyces sp. hsn06, as an actinomycete, can harvest C. vulgaris biomass safely and efficiently through flocculation activity, and proteins contribute greatly to the flocculation effect. However, potential flocculation protein-related genes are unclear. The mycelia of strain hsn06 after culture with glucose as the sole carbon source exhibited significantly higher flocculation activity as well as higher protein contents than those cultured with starch as the carbon source. To further explore the flocculation mechanism, the mycelia of strain hsn06 with distinct flocculation activities after culture with different carbon sources were examined by transcriptome analysis. We found that 403 genes were differentially up-regulated in mycelia cultured with glucose, compared to those cultured with starch as the carbon source. Five significantly differentially expressed protein-related genes were determined and confirmed by qRT-PCR, which indicated that three of the selected genes were potential flocculation-related genes. These results advance our understanding of potential flocculation-related genes during the harvesting of microalgal biomass.

Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395.

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

Effect of mixed culture of yeast and microalgae on acetyl-CoA carboxylase and Glycerol-3-phosphate acyltransferase expression.

In recent years, some studies have reported that co-culturing green algae and yeast improve lipid and biomass concentration. In this study, a co-culture of the oleaginous yeast Rhodotorula glutinis and the microalgae Chlorella vulgaris was consequently conducted with inoculation of microalga and yeast in growth and stationary phases, respectively. For the first time, the expression of two pivotal enzymes in fatty acids synthetic pathway, acetyl-CoA carboxylase and Glycerol-3-phosphate acyltransferase, was evaluated. To evaluate the synergistic impacts of the mixed culture on the enzymes expression, several co-culture models were designed, including the use of different ratio of microalgae to yeast or the use of residual cell-free medium of yeast; a positive impact on enzymes overexpression was shown in the case of the co-culture of the two microorganisms, and when the remaining cell-free medium of yeast was added to the microalgal culture. The results of in vitro co-culture demonstrated increased 6- and 5-fold of nervonic acid (C24:1) and behenic acid (C22:0) concentrations, respectively, in 2:1 microalgae to yeast co-culture as compared to the monoculture batches. Addition of yeast residual cell-free medium in the 2:1 ratio to the microalgal culture enhanced 9 and 6 times nervonic acid (C24:1) and behenic acid (C22:0) amounts, respectively.

Impacts of Endocrine Disruptor di-n-Butyl Phthalate Ester on Microalga Chlorella vulgaris Verified by Approaches of Proteomics and Gene Ontology.

Di-n-butyl phthalate (DBP) is an extensively used plasticizer. Most investigations on DBP have been concentrated on its environmental distribution and toxicity to humans. However, information on the effects of plasticizers on algal species is scarce. This study verified the impacts of endocrine disruptor di-n-butyl phthalate ester on microalga Chlorella vulgaris by approaches of proteomics and gene ontology. The algal acute biotoxicity results showed that the 24h-EC50 of DBP for C. vulgaris was 4.95 mg L-1, which caused a decrease in the chlorophyll a content and an increase in the DBP concentration of C. vulgaris. Proteomic analysis led to the identification of 1257 C. vulgaris proteins. Sixty-one more proteins showed increased expression, compared to proteins with decreased expression. This result illustrates that exposure to DBP generally enhances protein expression in C. vulgaris. GO annotation showed that both acetolactate synthase (ALS) and GDP-L-fucose synthase 2 (GER2) decreased more than 1.5-fold after exposure to DBP. These effects could inhibit both the valine biosynthetic process and the nucleotide-sugar metabolic process in C. vulgaris. The results of this study demonstrate that DBP could inhibit growth and cause significant changes to the biosynthesis-relevant proteins in C. vulgaris.

The establishment of new protein expression system using N starvation inducible promoters in Chlorella.

Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.

Augmenting the expression of accD and rbcL genes using optimized iron concentration to achieve higher biomass and biodiesel in Chlorella vulgaris.

Chlorella vulgaris is a form of microalgae commonly employed as a biological source of oil for biodiesel production. Major algal cultivation strategies are focused on stimulating growth rate and lipid content. In the present study, the algal growth media was supplemented with iron (III) chloride (FeCl3), as a stimulating factor for growth and lipid production, in three iron concentrations including 90, 200, and 500 µM. The turbidity of algal cells was measured on different days, to determine the growth rate. In optimum iron concentration, this measurement experienced a 2.1-fold increase. Next, the lipid content was extracted, and the amount of lipid produced in each treatment was calculated, which demonstrated a 4.57-fold increase in lipid productivity. The expression of genes corresponding to the metabolic enzymes (i.e. acetyl-CoA carboxylase (accD) and ribulose bisphosphate carboxylase large chain (rbcL)) was evaluated using real-time PCR under different initial iron feeds. As demonstrated in the results, the initial iron feed of 90 µM was an optimum concentration that obtained the highest growth rate, more cell density, and increased lipid production. In 90 µM initial iron concentration, the expression of accD and rbcL genes showed a 4.8- and 35-fold increase, respectively, compared to that of the control genes. Based on the results, this optimum iron concentration could satisfy the industrial interest in biodiesel production from C. vulgaris as a potential stimulating factor. However, higher levels of iron (e.g. 200 and 500 µM) failed to act as positive stress for increasing biodiesel production. Finally, in this paper, different mechanisms where iron affects acetyl-CoA carboxylase (ACCase) and 1,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCo) are illustrated.

Sulfonamides-induced oxidative stress in freshwater microalga Chlorella vulgaris: Evaluation of growth, photosynthesis, antioxidants, ultrastructure, and nucleic acids.

Sulfadiazine (SD), sulfamerazine (SM1), and sulfamethazine (SM2) are widely used and disorderly discharged into surface water, causing contamination of lakes and rivers. However, microalgae are regard as a potential resource to alleviate and degrade antibiotic pollution. The physiological changes of Chlorella vulgaris in the presence of three sulfonamides (SAs) with varying numbers of -CH3 groups and its SA-removal efficiency were investigated following a 7-day exposure experiment. Our results showed that the growth inhibitory effect of SD (7.9-22.6%), SM1 (7.2-45.9%), and SM2 (10.3-44%) resulted in increased proteins and decreased soluble sugars. Oxidative stress caused an increase in superoxide dismutase and glutathione reductase levels but decreased catalase level. The antioxidant responses were insufficient to cope-up with reactive oxygen species (hydrogen peroxide and superoxide anion) levels and prevent oxidative damage (malondialdehyde level). The ultrastructure and DNA of SA-treated algal cells were affected, as evident from the considerable changes in the cell wall, chloroplast, and mitochondrion, and DNA migration. C. vulgaris-mediated was able to remove up to 29% of SD, 16% of SM1, and 15% of SM2. Our results suggest that certain concentrations of specific antibiotics may induce algal growth, and algal-mediated biodegradation process can accelerate the removal of antibiotic contamination.

Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation.

Microalgae biorefinery is an alternative, sustainable and promising trend to solve the problem of fossil oil depletion and carbon dioxide emission. However, considering the innate limitation of cell growth and oil content in microalgae, to accelerate metabolic balance by CRISPR/Cas9 system is attractive. At first, plasmid based from Agrobacterium tumefaciens and a fragment of mGFP was transformed into Chlorella sorokiniana and Chlorella vulgaris FSP-E by electroporation, respectively. Selected colonies were tested by spectrophotometer and inverted fluorescence microscopy (IFM), and an increase of fluorescent was observed by 67% compared with that in wild type, which proved the Agrobacterium-mediated plasmid is suitable for gene insertion in Chlorella species. Consequently, plasmid with similar structure as mentioned previously containing fragment of Cas9 with sgRNA designed on omega-3 fatty acid desaturase (fad3) gene was constructed and showed a higher accumulation of lipid content by 46% (w/w) in C. vulgaris FSP-E. This is first-time to use CRISPR/Cas9 based technology for gene manipulation in Chlorella.

Early Stage Adaptation of a Mesophilic Green Alga to Antarctica: Systematic Increases in Abundance of Enzymes and LEA Proteins.

It is known that adaptive evolution in permanently cold environments drives cold adaptation in enzymes. However, how the relatively high enzyme activities were achieved in cold environments prior to cold adaptation of enzymes is unclear. Here we report that an Antarctic strain of Chlorella vulgaris, called NJ-7, acquired the capability to grow at near 0 °C temperatures and greatly enhanced freezing tolerance after systematic increases in abundance of enzymes/proteins and positive selection of certain genes. Having diverged from the temperate strain UTEX259 of the same species 2.5 (1.1-4.1) to 2.6 (1.0-4.5) Ma, NJ-7 retained the basic mesophilic characteristics and genome structures. Nitrate reductases in the two strains are highly similar in amino acid sequence and optimal temperature, but the NJ-7 one showed significantly higher abundance and activity. Quantitative proteomic analyses indicated that several cryoprotective proteins (LEA), many enzymes involved in carbon metabolism and a large number of other enzymes/proteins, were more abundant in NJ-7 than in UTEX259. Like nitrate reductase, most of these enzymes were not upregulated in response to cold stress. Thus, compensation of low specific activities by increased enzyme abundance appears to be an important strategy for early stage cold adaptation to Antarctica, but such enzymes are mostly not involved in cold acclimation upon transfer from favorable temperatures to near 0 °C temperatures.

Utilizing genome-scale models to optimize nutrient supply for sustained algal growth and lipid productivity.

Nutrient availability is critical for growth of algae and other microbes used for generating valuable biochemical products. Determining the optimal levels of nutrient supplies to cultures can eliminate feeding of excess nutrients, lowering production costs and reducing nutrient pollution into the environment. With the advent of omics and bioinformatics methods, it is now possible to construct genome-scale models that accurately describe the metabolism of microorganisms. In this study, a genome-scale model of the green alga Chlorella vulgaris (iCZ946) was applied to predict feeding of multiple nutrients, including nitrate and glucose, under both autotrophic and heterotrophic conditions. The objective function was changed from optimizing growth to instead minimizing nitrate and glucose uptake rates, enabling predictions of feed rates for these nutrients. The metabolic model control (MMC) algorithm was validated for autotrophic growth, saving 18% nitrate while sustaining algal growth. Additionally, we obtained similar growth profiles by simultaneously controlling glucose and nitrate supplies under heterotrophic conditions for both high and low levels of glucose and nitrate. Finally, the nitrate supply was controlled in order to retain protein and chlorophyll synthesis, albeit at a lower rate, under nitrogen-limiting conditions. This model-driven cultivation strategy doubled the total volumetric yield of biomass, increased fatty acid methyl ester (FAME) yield by 61%, and enhanced lutein yield nearly 3 fold compared to nitrogen starvation. This study introduces a control methodology that integrates omics data and genome-scale models in order to optimize nutrient supplies based on the metabolic state of algal cells in different nutrient environments. This approach could transform bioprocessing control into a systems biology-based paradigm suitable for a wide range of species in order to limit nutrient inputs, reduce processing costs, and optimize biomanufacturing for the next generation of desirable biotechnology products.

Transcriptome analysis of gene expression in Chlorella vulgaris under salt stress.

Chlorella vulgaris is an important freshwater alga that is widely used as a food source for humans and animals. High-salinity environments can cause accumulation of lipids and proteins in this species, but the mechanism of this accumulation and the salt response remain unclear. In this work, transcriptome analysis was performed for the C. vulgaris response to salt stress (1% and 3% NaCl) applied for different times (2 h and 4 h). In total, 5232 and 9196 were differentially expressed after 1% NaCl for 2 and 4 h, and 3968 and 9035 unigenes were differentially expressed after 3% NaCl for 2 and 4 h, respectively. The number of upregulated genes after 4 h of salinity stress was greater than the number of downregulated genes, suggesting that the alteration of gene expression may be related to a mechanism of adaptation to a high-salinity environment. Furthermore, gene ontology and KEGG pathway analyses revealed that numerous biological pathways are affected by salt stress. Among the upregulated pathways, the cytoplasmic calcium signaling pathway, which is involved in the regulation of homeostasis, was highly upregulated. Genes involved in the photosystem I light-harvesting pathway were downregulated under salt stress. These results provide foundational information on the effects of salt stress on C. vulgaris metabolism and its possible mechanism of surviving high concentrations of NaCl.

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

Chlorella vulgaris is a fast-growing fresh-water microalga cultivated on the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed us to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light, HL versus low light, LL) enabled us to identify 10 724 nuclear genes, coding for 11 082 transcripts. Moreover, 121 and 48 genes, respectively, were found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed particular features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL versus HL provided insights into the molecular basis for metabolic rearrangement under HL versus LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway could be predicted and its upregulation upon HL exposure was observed, consistent with the increased lipid amount under HL conditions. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.