We have learned that the cyanobacteria can be degraded efficiently by sophorolipid from some papers. So the cyanobacteria degradation experiment was carried out to verify its effect.
Pretest study: Determination of the appropriate content of cyanobacteria
The risen cyanobacteria were collected and were acting as the algae mother liquor, which had a concentration of 0.936 g/mL. Three groups were divided by diluting the mother liquor twice, 10 times and 20 times, respectively. Add equal amount of sophorolipids powder to the test beakers and mix up to the final content of 20 mg/L. Then observe the degradation effect of different groups.
After 5 minutes, the cyanobacteria in group 3 have been degraded almost totally. There was also obvious phenomenon in group 1 and group 2. After 2 hours, most of the cyanobacteria have been degraded.
Summary: To achieve the adequate reaction time, the group 1 is the best choice.
Fig.1. Degradation effect of cyanobacteria after 5 minutes
Degradation process: Find a preliminary method for determining the degradation efficiency
Dilute the mother liquor 10 times and add them to two centrifuge tubes. Add 2 mL sophorolipid with a concentration of 15 mg/mL to one centrifuge tube as the test group, while the control group added equal deionized water. Shake well and then observe the degradation process. After 15 min, these two tubes were centrifuged at 7000 RPM for 2min, and then observe the phenomenon.
The cyanobacteria have been degraded totally after adding sophorolipid with a high concentration. After centrifuging, the dead cyanobacteria settled down into precipitate, while the live cyanobacteria floated on the top.
Video1. Cyanobacteria degradation process
Summary: Observing the precipitation and rising cyanobacteria after centrifugation can be a preliminary method for determining the degradation efficiency.
Best ratio: Test the best ratio that can maximize the degradation of cyanobacteria.
Dilute the mother liquor twice and add them to eight centrifuge tubes. Then we mixed two kinds of pure sophorolipids (both acid and lactone with the concentration of 0.991 mg/mL) to obtain mixed sophorolipids with different ratios shown in the table below. 10 μL of these combinations were added to the corresponding tubes, respectively. The degradation efficiency was measured by preliminary centrifugation observation and testing the chlorophyll content of living cells.
Observe the amount of precipitate and rising cyanobacteria. In Fig.2, Lactone : Acid ratio of 10 : 0, 8 : 2 and 6:4 seem to have the most precipitate and least rising cyanobacteria.
Fig.2. Preliminary concentration observation
Chlorophyll in living cells was extracted with acetone. Test the absorbance of different groups at the λ=663 and λ=645 and note them as E663 and E645. Then the relative chlorophyll content can be calculated by the formula “10.115×E663-1.877×E645”.
Tab.1. The relative chlorophyll content
Summary: The lactone : acid SL ratio of 8:2 has the highest efficiency on cyanobacteria degradation.
Fig.3. The degradation efficiency of cyanobacteria
Our team intends to use strong promoter to express Cas9 protein and sgRNA and use different promoters to regulate different ratios of acid and lactone sophorolipids via regulating the expression level of key gene. Therefore, we need to identify the intensities of different promoters screened before constructing the recombinant strains producing sophorolipids in this work.
Construction of GFP expression plasmid: Expression of GFP in Starmerella bombicola.
We constructed GFP expression cassette and transformed it into S. bombicola to express GFP. After incubation, observe the fluorescence (Fig 4).
Fig.4. Analysis of GFP expression in S. bombicola
GFP can be successfully expressed in Starmerella bombicola.
Promoter screening: Determination of the promoter intensity
We obtained 14 different promoters from S. bombicola and identified their functions. These promoters were used to direct the expression of GFP protein in S. bombicola. We found that the effect of these promoters on GFP expression were different, and the intensity of these promoters was also different.
From the Fig.5. below, we can know the intensity of these 14 promoters.
Fig.5.Analysis promoters’ intensity by fluorescence observation and transcription level analysis
Summary: Ptef1 is the strongest promoter that can be used to express sgRNA and Cas9 protein.
As an efficient gene editing system, CRISPR/Cas9 system was used to insert the promoters screened into specific sites of S. bombicola genome to regulate the ratio of acid and lactone sophorolipids in this work.
NLS nuclear localization signal peptide:Guide Cas9 through the nucleus.
Since the molecular weight of Cas9 nuclease is high, Cas9 nuclease cannot freely pass through the nuclear pores. We need to add a nuclear localization sequence (NLS) to guide it through the nucleus. Under the guidance of sgRNA, Cas9 nuclease can cut the specific sites of genome to form the double strains breaks (DSBs), and then the target gene could be engineered.
We linked the optimized NLS sequence to the 3' end of the gene gfp to construct an expression plasmid, and then transformed it into S. bombicola. The positive colonies were screened by detecting the green fluorescence intensity of GFP (Fig.6). We found that the luminescent region was concentrated in the nucleus. According to the results, it was concluded that the signal peptide can be used for the subsequent construction of the CRISPR/Cas9 system in S. bombicola.
Fig.6 The effect of the signal peptide on the subsequent construction of the CRISPR/Cas9 system in S. bombicola.
Without signal peptide (Left) and with signal peptide (Right)
The optimized NLS can successfully guide Cas9 nuclease through the nucleus to cut the specific sites.
Determination of gene editing efficiency: Verifying the feasibility of CRISPR/Cas9 gene editing system
We carried out the verification experiments of single, double and triple gene-editing efficiency. We firstly integrated the Cas9 expression cassette into the genome as a cutting tool for gene editing, and then used the colonies containing Cas9 nuclease to verify the subsequent editing efficiency.
√ Single gene-editing efficiency
We constructed the recombinant fragment PXA1-hphex-PXA1-sgPXA1-13ex and transformed it into S. bombicola, which achieved the knockout of PXA1 gene and inserted the hygromycin resistant gene as a selection marker. The transformants were spread on YPD plus hygromycin plates. Then the positive transformants were conducted genome extraction for PCR and gel electrophoresis analysis. As a result, all of the amplified fragments displayed the correct stripe. So all of the target genes were verified to be inserted into the Pxa1 site.
The single gene-editing efficiency was
Fig.7 Gel electrophoresis analysis of the positive transformants
√ Double gene-editing efficiency
Secondly, we inserted GFP expression cassette into the S. bombicola genome, and obtained the positive colonies by analysing green fluorescence intensity. The positive colonies can be used as the starting strains to verify the double-gene editing efficiency. We constructed the expression fragment PXA1-hphex-PXA1-sgPXA1-13ex-linker-sg-GFP-GFP-donor, which realized the knockout of GFP genes and inserted the hygromycin resistant gene. The colonies were screened on the solid medium with hygromycin (Fig.8, Left), which also lost the function of expressing GFP (Fig.8, Right). It was verified that the double gene-editing of CRISPR/Cas9 system in S. bombicola was successful in this work.
The double gene-editing efficiency was
Fig. 8. Double-gene editing efficiency of CRISPR/Cas9 system in S. bombicola
√ Triple gene-editing efficiency
We also identified a gene related to synthesis of leucine, and used it as the target gene to verify the editing efficiency of CRISPR/Cas9 system in S. bombicola. It was verified that the triple-gene editing efficiency of CRISPR/Cas9 system in S. bombicola was high using the leucine synthesis gene, PXA1, and gfp (Fig9).
The triple gene-editing efficiency was
Fig.9. Triple-gene editing efficiency of CRISPR/Cas9 system in S. bombicola
Medium with leucine (Left) and minimal medium without leucine (Right)
Single gene-editing efficiency can up to 100%, double one can up to 99% and triple one can up to 30%.
Summary: Successfully construct CRISPR/Cas9 system with high gene-editing efficiency in S. bombicola.
Production of Sophorolipids
Pure acid type and lactone type sophorolipids
The strong promoters were used to express UGTB, and as a result, the production of sophorolipids had increased. Starting with the high production-level strains, we knocked out SBLE to produce pure acid-type sophorolipid and over-expressed SBLE with strong promoters to produce pure lactone-type sophorolipid.
The yield had boosted a lot by over-expressing UGTB. The pure acid-type and lactone-type sophorolipid had also been produced (Fig.10).
Fig.10. Sophorolipids yield