Team:Worldshaper-Nanjing/Proof Of Concept
1 Introduction
For the purpose of converting gutter oil into γ-linolenic, we strengthen the oil metabolism of Yarrowia lipolytica ( Y. lipolytica ) and give it the ability to synthesize γ-linolenic by overexpressing the β-oxidation pathway and introducing the Δ6-fatty acid desaturase in the Y. lipolytica po1f. The reasons and details for introducing these biobricks were written on the Design page. In order to confirm the potential of the transgenic Y. lipolytica po1f to metabolize waste oil, we made further fermentation verification.
2 Testing the abilities of engineering yeasts for utilizing the edible oil
2.1 Yeast transformation
We constructed 6 multi gene combined expression plasmids by combining β-oxidation pathway genes (ylPOT1, ylMFE1, and ylPOX1-6) together on plasmid pYLXP’ , and assembled Δ6-fatty acid desaturase gene M-Δ6-D into the hp4d-based expression vector p0. Then all of the plasmids (pYXLP’-ylPOT1-ylMFE1-ylPOXn and p0-6-D, Table1) were tranformed into Y. lipolytica po1f based on the standard protocols of Y. lipolytica transformation by the lithium acetate method.
In brief, one milliliter cells was harvested during the exponential growth phase (16-24 h) from 2 mL YPD medium (yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L) in the 14-mL shake tube, and washed twice with 100 mM phosphate buffer (pH 7.0). Then, cells were resuspended in 105 uL transformation solution, containing 90 uL 50% PEG4000, 5 uL lithium acetate (2M), 5 uL boiled single stand DNA (salmon sperm, denatured) and 5 uL DNA products (including 200-500 ng of plasmids, lined plasmids or DNA fragments), and incubated at 39 ℃ for 1 h, then spread on selected plates. The selected marker is leucine in this project. The results of transformation have been showed in Figure1 .
Table1 The recombined plasmids
Fig.1 The plates of Y. lipolytica po1f transformation for overexpressing β-oxidation pathway
2.2 Shaking flask cultivations for testing the abilities of engineering yeast to utilize the edible oil
For performing shake flask cultivations, seed culture was carried out in the shaking tube with 2 mL seed culture medium at 30 ℃ and 250 r.p.m. for 48 h. Then, 0.8 mL of seed culture was inoculated into the 250 mL flask containing 30 mL of fermentation medium and grown under the conditions of 30 ℃ and 250 r.p.m. for 120 h. One milliliter of cell suspension was sampled every 24h for OD600 measurements. The results of Time profiles of cell growth of engineering strains have been showed in Figure2 and Figure 3. The experimental results showed that combined overexpression of genes ylPOT1, ylMFE1, and ylPOX4 or ylPOX5 (engineering strains po1f-pYLXP’-ylPOT1-ylMFE1-ylPOX4 and po1f-pYLXP’-ylPOT1-ylMFE1-ylPOX5) significantly improve the cell growth of Y. lipolytica with using the edible oil as the substrate.
Seed culture medium used in this study included the yeast complete synthetic media regular media (CSM, containing glucose 20.0 g/L, yeast nitrogen base without ammonium sulfate 1.7 g/L, ammonium sulfate 5.0 g/L, and CSM-Leu 0.74 g/L) and complex medium (YPD, containing glucose 20.0 g/L, yeast extract 10.0 g/L, and peptone 20.0 g/L). Fermentation medium used in this study also included the yeast complete synthetic media regular media (CSM, containing glucose 40.0 g/L, yeast nitrogen base without ammonium sulfate 1.7 g/L, the edible oil 75 ml/L, and CSM-Leu 0.74 g/L).
Fig.2 Time profiles of cell growth of engineering strains
Fig.3 Seed culture and shaking flask cultivation of engineering strains
3 Producing γ-linolenic acid with using the edible oil by engineering yeasts
3.1 Genomic integration of gene M-Δ6-D into the improved strains
We transformed the recombinant vector p0-6-D that containg Δ6-fatty acid desaturase gene M-Δ6-D to the improved strains (engineering strains po1f-pYLXP’-ylPOT1-ylMFE1-ylPOX4 and po1f-pYLXP’-ylPOT1-ylMFE1-ylPOX5) and obtained engineering strains po1f-pYLXP’-ylPOT1-ylMFE1-ylPOX4-M-Δ6-D and po1f-pYLXP’-ylPOT1-ylMFE1-ylPOX5-M-Δ6-D. The selected marker is uracil, and he positive transformants were determined by colony PCR, and the result has been showed in Figure 4 and Figure 5.
Fig.4 The plates of Y. lipolytica po1f transformation for introducing gene M-Δ6-D
Fig.5 The positive transformants were determined by colony PCR. (a) po1f-pYLXP’-ylPOT1-ylMFE1-ylPXO4-M-Δ6-D; (b) po1f-pYLXP’-ylPOT1-ylMFE1-ylPXO5-M-Δ6-D
3.2 Shake flask cultivations for producing γ-linolenic acid
The procedure of genomic integration of shake flask cultivations was described above. The result has been showed in Figure 6 and Figure 7. One milliliter of cell suspension was sampled every 24h for γ-linolenic acid measurements. Specifically, we obtained a titer of 59.3 mg/L γ-linolenic acid produced by the engineered strain po1f-pYLXP’-ylPOT1-ylMFE1-ylPOX4-M-Δ6-D with using the gutter oil as substrate, which significantly higher than the starting strain.
Fig. 6 Gas chromatogram analysis of γ-linolenic acid titer in engineering strains
Fig. 7 Time profiles of engineering strains to produce γ-linolenic acid
4 Future work
Due to the epidemics of Covid-19, several following plans have not been able to carry out, as the experimental time of this project is limited. According to the experimental results, we are ready to further test our yeast’s efficiency of converging gutter oil instead of edible oil to γ-linolenic acid. We could then use these data to expand the fermentation system in order to achieve the requirements of industrial production. In this way, we can better realize the sustainable development goals of protecting the global environment and waste utilization.