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Overview

Our results consist of three main parts: Firstly, confirmation of optimal growth conditions of Chlamydomonas reinhardtii. Secondly, observation and transcriptome analysis of Chlamydomonas reinhardtii under different stress conditions. Thirdly, genetic engineering of Chlamydomonas reinhardtii by using CRISPR/Cas9 system.

1 Confirmation of optimal growth conditions of Chlamydomonas reinhardtii

1.1 Basic growth curve

To confirm the culture pattern of Chlamydomonas reinhardtii in our lab, we measured the absorbance values of Chlamydomonas reinhardtii at 750nm every 12h to obtain the growth curve of Chlamydomonas reinhardtii. Then, to further confirm the relationship between absorbance values at 750nm and cell number, we used a hemocytometer plate to count Chlamydomonas reinhardtii with different absorbance values and obtained the corresponding formula between absorbance values and cell numbers.

Figure 1.Growth curve and fitted curve of Chlamydomonas reinhardtii.
(A)Growth curve of Chlamydomonas reinhardtii.
(B)Fitted curve of Chlamydomonas reinhardtii.

Before 36h, Chlamydomonas reinhardtii grew rapidly (Figure 1A). After that, the growth of Chlamydomonas reinhardtii reached a plateau. Also, there was a correlation between the absorbance value at 750nm and cell number in Chlamydomonas reinhardtii (Figure 1B).

1.2 Optimization of the aseptic culture system

Keeping sterile is fundamental to microalgae biotechnology as well as basic scientific research. However, contamination by bacteria and/or fungi in microalgae cultures is often a triky problem.

Amp is the most commonly used antibiotic for bacterial inhibition when culturing Chlamydomonas reinhardtii. However, in the process of cultivation, even if we used Amp with a concentration three times higher than that used in the paper, the pollution of Chlamydomonas reinhardtii culture still remains, which seriously affects the accuracy of experimental data.

To solve this problem, we selected two other antibiotics commonly used in culturing plants: Cef and Tim, and carried out exexperimental research.

Figure 2.Growth and contamination of Chlamydomonas reinhardtii after 3 days of culture in TAP medium which contained different graded concentrations of Cef, Tim and Amp.
(A)Absorbance values of Chlamydomonas reinhardtii at 750nm.
(B)Contamination of Chlamydomonas reinhardtii after centrifugation.

The experimental results showed that the use of TAP medium containing Tim had little effect on the growth of Chlamydomonas reinhardtii (Figure 2B), and the use of Tim at a concentration of 250μg/mL resulted in the least contamination (Figure 2A). Therefore, we decided to use this condition to replace previously used 300μg/mL Amp to cultivate Chlamydomonas reinhardtii so as to reduce the contamination of miscellaneous bacteria under normal culture conditions.

It is worth noting that Tim was applied to cultivate Chlamydomonas reinhardtii for the first time. Here, we provide a reference for other teams that take Chlamydomonas reinhardtii as the chassis organism to select the conditions for cultivation, which enables them to obtain more accurate experimental data.

2 Identification of key genes promoting lipid production in Chlamydomonas reinhardtii

2.1 Culture and analysis of Chlamydomonas reinhardtii under different stress treatments

2.1.1 Selection of stress treatments

Nutrition deficiency, temperature and light are important factors to regulate lipid production in Chlamydomonas reinhardtii, among which nutrition deficiency caused by stress treatment has a significant effect on promoting lipid production.

Nitrogen and its compounds play an important role in regulating gene expression and metabolic pathways in Chlamydomonas reinhardtii. Nitrogen stress can significantly promote lipid production, and it is the most studied and effective stress treatment ever. Iron is an essential micronutrient for all organisms and plays an important role in the growth and metabolism of Chlamydomonas reinhardtii. Studies showed that severe Iron stress can lead to the accumulation of different types of lipids in algal cells, especially triacylglycerol (TAGs), under which condition the initial growth of Chlamydomonas reinhardtii will not be strongly inhibited. Although Iron stress is relatively poorly studied in Chlamydomonas reinhardtii, it is still a very promising treatment for promoting its lipid production.

Therefore, we chose conventional Nitrogen stress and novel Iron stress among various stress treatments to observe their effects on growth and lipid production of Chlamydomonas reinhardtii.

2.1.2 Effects on growth and lipid production of Chlamydomonas reinhardtii under different stress treatments

We cultured Chlamydomonas reinhardtii in TAP under Nitrogen and Iron stress respectively for 48h and performed three biological replicates to ensure data accuracy.

To explore the effects on growth of Chlamydomonas reinhardtii under different stress treatments, we measured the absorbance value at 750nm on a regular basis during the culture process and used the obtained basic growth curve for comparison.

To explore the effects on lipid production of Chlamydomonas reinhardtii under different stress treatments, we used fluorescence microscopy to qualitatively detect the lipid content and used Image J to quantitatively analyze the lipid production status. For the staining method, we chose Nile red staining, which is the most commonly used method for rapid detection of lipid content in microalgae.

Figure 3. Growth and lipid production of Chlamydomonas reinhardtii under different stress treatments.
(A) Fluorescence microscopy image. G light (492nm-577nm) excitation, 80ms exposure time, 400 sensitivity.
(B) Growth curves of the control group(TAP), Nitrogen stress group, and Iron stress group.
(C) Average fluorescence intensity analysis.

In the early stage, there was no significant difference between Nitrogen stress group and the control group, but gradually the growth rate of Nitrogen stress group got lower than that of the control group, indicating that Nitrogen stress treatment had a certain inhibitory effect on the growth of Chlamydomonas reinhardtii. However, the growth rate of Iron stress group was constantly higher than that of the control group, indicating that Iron stress treatment did not limit the growth of Chlamydomonas reinhardtii (Figure 3B).

The average lipid content of the control group did not change with time. At 8h, the average lipid content of Nitrogen stress group and Iron stress group was 142.3% and 194.8% of that of the control group respectively. At 24h, the average lipid content under Nitrogen stress group and Iron stress group was 259.7% and 232.7% of that of the control group respectively. To conclude, in the process of culture, Iron stress treatment had a small increase in the average lipid production, while Nitrogen stress treatment showed a significantly better result than the other two groups (Figure 3C). The fluorescence microscopy images showed consistent findings (Figure 3A).

2.2 Transcriptome sequencing analysis

The environment lacking nutrients or essential mineral factors would cause algal cells to undergo a series of physiological reactions, which involve the differential expression of a series of genes. To minimize environmental differences, we chose to compare Nitrogen stress group and Iron stress group to identify genes that may affect lipid production. Previous experiments had shown that the growth of Nitrogen stress group and Iron stress group began to differ at 8h. In addition, the lipid production of Nitrogen stress group and Iron stress group was significantly different at 24h. Therefore, we sampled Chlamydomonas reinhardtii cultured for 8h and 24h respectively under both Nitrogen stress treatment and the Iron stress treatment for transcriptome sequencing.

2.2.1 Sequencing data statistics

The raw data from Illumina HiSeq sequencing was filtered to obtain the initial data of four samples, 28.31Gb clean reads in total. The percentage of Q30 bases in each sample was more than 93.14%, indicating that the error probability of base recognition was low (Table 1).

Table 1. Sequencing data statistics.


We obtained the total gene expression of each sample (Click here know more). FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) is often used as a measure of transcript or gene expression level.

2.2.2 Differential gene expression analysis

Gene expression is time-specific and space-specific. We performed differential expression analysis of transcripts under four different conditions and obtained differential gene expression results in pairs (Table 2). Click here know more.

Table 2. The calculation of differential expression genes.


Between the comparison of group N8h_vs_Fe8h and group N24h_vs_Fe24h, about 72% of all 5398 transcripts were differentially expressed (criteria: Fold Change≥2, FDR＜0.05), which is consistent with previous studies showing that Nitrogen or Fe stress treatment induces extensive transcriptional changes related to lipid production. Considering that the total lipid production under Nitrogen stress treatment and Fe stress treatment had obvious differences over time, we made an in-depth analysis of the reasons for the difference in the total lipid production between these two groups.

2.2.3 Enrichment analysis of differential gene expression

KEGG pathway-based analysis (Figure 4) revealed that differential expressed genes were mainly enriched in "Fatty acid biosynthesis", "Fatty acid metabolism", "Nitrogen metabolism" and "Valine, leucine and isoleucine degradation". Consequently, we did specific analysis of these four pathways.

Figure 4. KEGG enrichment map of differential expressed genes.
(A) N8h_vs_Fe8h KEGG enrichment map of differentially expressed genes.
(B) N24h_vs_Fe24h KEGG enrichment map of differentially expressed genes.

2.2.4 Cluster analysis of differential genes by thermography

Hierarchical cluster analysis was conducted on the differentially expressed genes screened from the above pathways, and the genes of the same metabolic pathway were clustered to form thermography of the clustering results of differentially expressed genes. Consequently, the target genes that significantly increased the lipid content of Chlamydomonas reinhardtii were selected.

Figure 5. Fatty acid biosynthesis clustering thermography.

Based on the analysis of KEGG database, 25 genes were annotated into the fatty acid pathways for synthesis, of which 13 genes were significantly different in the two control groups. We did the rankings according to the discrepant degree under N and Fe stress conditions in 8h and 24h respectively, thus selecting the 3 genes with the greatest differential degree (Figure 5).

Figure 6. Fatty acid metabolism clustering thermography

Based on the analysis of KEGG database, 40 genes were annotated into metabolism pathway of fatty acid, of which 18 genes were significantly different in the two control groups. We did the rankings according to the discrepant degree under N and Fe stress conditions in 8h and 24h respectively, thus selecting the 2 genes with the greatest differential degree (Figure 6).

Figure 7. Nitrogen metabolism clustering thermography.

Based on the analysis of KEGG database, 18 genes were annotated into the nitrogen metabolism pathway, of which 9 genes were significantly different in the two control groups. We did the rankings according to the discrepant degree under N and Fe stress conditions in 8h and 24h respectively, thus selecting the 4 genes with the greatest differential degree (Figure 7).

Figure 8. Valine,leucine and isoleucine degradation clustering thermography

Based on the analysis of KEGG database, 20 genes were annotated to metabolic pathways valine,leucine and isoleucine degradation, among which 113 genes were significantly different in the two control groups. We did the rankings according to the discrepant degree under N and Fe stress conditions in 8h and 24h respectively, thus selecting the 2 genes with the greatest differential degree (Figure 8).

Table 3. Potential genes to promote lipid production in the four metabolic pathways.


To sum up, based on the amount of expression, we summarized the differential expression of four pathways, and selected 12 genes with high/low expression (Table 3), which can be used as the sites for subsequent regulation genes to provide target sites for gene editing of lipid production in Chlamydomonas reinhardtii and the resources and information of genes for basic research and application research.

3 The strategy of using genetic engineering

3.1 The design and construction of CRISPR/Cas9 system

Based on transcriptome sequencing results and previously reported genes, we aim to improve lipid production in Chlamydomonas reinhardtii under non-stress conditions by using synthetic biology and gene editing methods.

We designed the CRISPR/Cas9 system for editing Chlamydomonas reinhardtii (Figure 9). In this system, we selected mCherry, which is the best available reporter gene in the plant field, and StayGold, a bright green fluorescent protein that has recently been shown to be highly photostable as our reporter genes and we optimized this two elements according to the codon preference of Chlamydomonas reinhardtii. At the same time, each vector carried an optional marker box, C. reinhardtii503 which could be granted resistance to Hyg. In the selection of promoters, we selected RBCS2 and HSP70A. For terminators, polyT Term and RBCS2 Term were selected.

We used Golden Gate Assembly and Gibson Assembly to construct each element into pTX2038 and pTX2040 vectors, and amplified and expressed them in E.coli DH-5α to obtain the basic skeleton vector. To verify the successful construction of the vector, PCR amplification was performed.

Figure 9. Structure of pTX2038 and pTX2040 plasmids and single colony PCR verification.
(A) The structure of pTX2038.The pTX2038 contains three main components: Cas9, mCherry and HgR.
(B) The structure of pTX2040.The pTX2040 contains three main components: Cas9, StayGold and HgR.
(C) Cas9, StayGold, HgR sequence fragments of pTX2038 and Cas9, StayGold, HgR sequence fragments of pTX2040 were successfully amplified by single colony PCR. M: 2000bp DNA Marker; Cas9, HgR, mCherry, StayGold: fragments of corresponding elements.

The brightness of the primer fragment amplified PCR product matched with the DNA Marker marker position (Figure 9C), which initially proved that the sgRNA of the target gene was inserted into the vector. Subsequently, we performed Sanger forward sequencing and sequence comparison for each insert site to further verify the successful vector construction.

3.2 Transformation

3.2.1 The determination of optimal screening concentration

According to the specificity of the codon of Chlamydomonas reinharditii, our team optimized and designed a new part for the ChlamyHgR (Part:BBa_K4335003) based on the existing part, and the new part acted as the selection standard for transforming positive clones. However, the inhibition effect of hygromycin varies with species. Therefore, we designed and performed experiments to determine the minimum inhibitory concentration of Hyg on the growth of Chlamydomonas reinhardtii in solid medium.

Figure 10. The wild type Chlamydomonas reinharditii CC-503 (2 x 108 cells) cells were placed on TAP solid medium supplemented with different concentrations of Hyg (10, 20 and 25µg/mL), and the growth of Chlamydomonas reinhardtii on the solid plate was continuously observed. The pictures displayed correspond to a 2-week old culture. The experiment was repeated in three copies.

After two weeks of incubation, we observed an inverse relationship between the number of colonies growing in the culture dish and the concentration of Hyg (Figure 10). Only in the plates with a concentration of 25µg/mL Hyg we consistently observed inhibition of cell growth. Therefore, we decided to use this concentration in TAP media to carry out the experiments described below.

3.2.2 The procedure of transformation

We chose to use the method of electrotransformation to transform Chlamydomonas reinhardtii. In order to improve the efficiency of electrictransformation, we used GeneArt® MAX Efficiency® Transformation Reagent for Algae (Therfisher, # A24229) to carry out the experiment.

1-2 x 108 cells of algal solution and 2-4μg of linear plasmids were used in each electrotransformation. The linear pTX2038 and pTX2040 vectors were respectively electroporated into Chlamydomonas reinhardtii strains at 635V, 31μF, and 800Ω. The cells were allowed to recover for 14-16 hours under the condition of continuous light and vibration treatment. Single colonies were separated on solid plates containing 25μg/mL Hygromycin. For detailed information, you can refer to our workflow(Figure11).

Figure 11. Workflow diagram summarizing steps for electrotransformation.

After 7 days, the resistant colonies which grew vigorously were obtained in the experimental group (Figure 12A), while no algae grew in the negative control group. The number of monoclone which had been successfully transferred into pTX2038 and pTX2040 in the plate was counted respectively and their transformation efficiencies was also calculated respectively (Figure 12B), after which process the editing efficiency of 1.9-2.1 x 10-6 was achieved, and a relatively effective transformation system of Chlamydomonas reinhardtii was established.

Figure 12. Selection and genetic editing efficiency of thaumatin-resistant colonies after transformation.
(A) Growth of Chlamydomonas reinhardtii in the plates after electrotransformation (7 days). Negative control: no plasmid was added. All dishes shown in the figure contain TAP medium supplemented with 25µg/mL of Hyg, except for the positive control of the wild-type Chlamydomonas reinhardtii strain.
(B) Statistics of positive clones after transformation ang the frequency of transformation.

3.2.3 Molecular test

To verify whether the transfer of the vector into 503 cells of Chlamydomonas reinhardtii at the molecular level is successful or not, we used the colony PCR method to ensure full integration of the vector.

Figure 13. Gel run of samples from colony PCR.
(A) Sequence comparison of Cas9, HgR, and mCherry fragments in pTX2038.
(B) Sequence alignment of Cas9, HgR, and StayGold fragments in pTX2040. M: 2000bp DNA marker; NC: wildtype; PC: Linear plasmid. 1-3 indicate different transformants selected.

The monoalgal colonies of pTX2038 and pTX2040 have been transferred. The brightness of PCR products of Hyg resistance gene, mCherry reporter gene, Cas9 protein and StayGold reporter gene (Figure 13) fragments were all consistent with the DNA bands of the positive control group as well as matched with the position of DNA Marker, which indicates that the fragmented PCR products transferred into the vector were in a high concentration and normal expression state. In sum, the effectiveness of transformation could be testified.

3.2.4 Fluorescent validation

To further demonstrate the correct insertion of the vector at the cellular expression level, we performed microscopy whose centers were the autofluorescent genes contained in the construct vectors: mCherry and StayGold. And autofluorescence in red of Chlorophyll was used as a negative control. It is worth noting that although both chlorophyll autofluorescence and mCherry will appear red after fluorescence excitation at certain wavelengths, they do not affect the observation because the wavelength range of their excitation of fluorescence does not overlap.

Figure 14. Images of WT Chlamydomonas reinhardtii and positive clones after transformation at 20x magnification. WT: wild type; DIC: bright field. 60ms exposure time; Chlorophyll: EX:Form 625nm to 650nm, 10ms exposure time; mCherry: EX:Form 515nm to 555nm, 300ms exposure time; StayGold: EX:Form 465nm to 495nm, 300ms exposure time.

Compared with the wildtype, the cells of Chlamydomonas reinhardtii with fluorescent reporter gene showed different degrees of fluorescence and normal autofluorescence expression of chlorophyll, which further proved that the vector could be expressed normally when transferred into Chlamydomonas reinhardtii (Figure 14).

3.3 The strategy of efficient lipid production for Chlamydomonas reinhardtii

3.3.1 Target gene selection

To promote lipid accumulation in Chlamydomonas reinhardtii, we based on existing reports to focus on blocking the decomposition of fatty acid, promoting the secretion of fatty acid out of the cellular, reducing the TCA cycle, reducing pathways of carbon flowing for biomass synthesis, blocking the initial stage of LD degradation, promoting the generation of the acyl glyceride, and regulating cell cycle to select a series of genes (Figure 15): ACX2 (NCBI; Gene ID:5727823), LACS1 (NCBI; Gene ID:5728486), LACS2 (NCBI; Gene ID:5716500), PEPC1 (NCBI; Gene ID:66056659), ICL1 (NCBI; Gene ID:5720950, DTH1 (NCBI; Gene ID:5722030, PLA2 (NCBI; Gene ID:5725622, CHT7 (NCBI;Gene ID:5722949).

Meanwhile, by transcriptome analysis, we selected a series of genes with the greatest degree of variation (Figure 15): 3-ACPR (Gene ID:gene-CHLRE_04g216950v5), ACC (Gene ID:gene-CHLRE_08g359350v5), BCCP (Gene ID:gene-CHLRE_17g715250v5), Acox (Gene ID:gene-CHLRE_11g467350v5), ω-6FAD (Gene ID:gene-CHLRE_17g711150v5), CYP450 family (Gene ID:gene-CHLRE_01g007950v5), CA (Gene ID:gene-CHLRE_04g223100v5), NRT (Gene ID:gene-CHLRE_09g396000v5), NiRs (Gene ID:gene-CHLRE_09g410750v5), DLD (Gene ID:gene-CHLRE_01g016514v5), KADHs (Gene ID:gene-CHLRE_06g311050v5), MCC (Gene ID:gene-CHLRE_03g181200v5).

Figure 15. The selected genes. The

blue

ones are reported genes and the

green

ones are the genes obtained by using transcriptome analysis.

3.3.2 sgRNA design

We used the tool called CRISPOR on the sgRNA design website. At the same time, combined with the off-target prediction model, the exons which have higher rankings were selected to design sgRNA, and the sequences with higher comprehensive scores were selected to design three sgRNA for each gene (Figure 16). For details about the primers we designed, please refer to the attachment prime.

Figure 16. Schematic diagram of respective sgRNA positions of some target genes (PLA2, ICL1, CHT7).

3.3.3 Vector construction

We used Golden Gate Assembly to insert the sgRNA fragments of each target gene into the pTX2038 and pTX2040 vectors, respectively, and amplified the expression in E. coli DH-5α to obtain a CRISPR/Cas9 system that could knock out the target sequence. In order to verify the successful construction of the vector, PCR amplification was performed by the forward of target gene and downstream primers iGEM007 (pTX2038) or iGEM008 (pTX2040). Please refer to the attachment prime for primer sequences.

Figure 17. Gel run of samples from colony PCR. Primer length ~660 bp. M: 2000 bp DNA Marker; 38: pTX2038 vector; 40: pTX2040 vector; sgRNA1-3: different sgRNAs are designed.

The brightness of amplified primer fragments matched the position of DNA Marker (Figure 17), which initially proved that the sgRNA of the target gene was inserted into the vector. Subsequently, Sanger forward sequencing and sequence alignment were performed for each insert site to further verify the successful construction of the vector.

3.3.4 Creation and validation of PSY1 mutants

To demonstrate the integrity of the introduced vector and the proper functioning of the CRISPR/Cas9 system, we chose to use the phytoene synthase gene PSY1 as the target gene, the knockout of which will result in the cell state of Chlamydomonas reinhardtii being white under microscopic bright-field conditions. We successfully aspirated parts of algal solutions amplified in 96-well plates for filming and observation in the bright field of the microscope, and used wild type as a negative control (Figure 18).

Figure 18. Comparison between wildtype Chlamydomonas reinharditii and transformed positive clones in bright field.

Here, we provide a scheme to verify the successful introduction and proper operation of the CRISPR/Cas9 system by targeting the endogenous PSY1 gene for vector construction and transformation, and by microscopic bright-field comparison of the wild type with the PSY1 mutant, which proves that the system is feasible if the field of view turns white.

4 Outlook

Based on the results, we confirmed the effective culture system and stress conditions. Meanwhile, we completed the microalgae culture under stress conditions and transcriptome analysis of Chlamydomonas reinhardtii. We also discovered a series of genes with a large degree of difference. In addition, we successfully constructed a CRISPR/Cas9 knockout system and a relatively mature transformation system for Chlamydomonas reinhardtii . The feasibility of which was verified by using PSY1 as a reporter gene.

In the future, the following work is projected to be done.
1. Molecular biological validation of these genes' functions in the process of lipid anabolic biology was performed for genes with a large degree of variation obtained from transcriptome analysis.
2. Complete the construction of all candidate gene mutants and evaluate their effects on the lipid production capacity of Chlamydomonas reinhardtii.
3. Improve the overall design of the applicable system and technology, and develop an integrated strategy for multi-gene.

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