Team:Worldshaper-Nanjing/Engineering
1 Overview
Y. lipolytica, as a ‘generally regarded as safe’ (GRAS) yeast, has been extensively engineered for the production of oleochemicals, fuels and commodity chemicals. In this project, to achieve the production of γ-linolenic acid with the gutter oil as substrate, we introduced the Δ6-fatty acid desaturase and overexpressed the β-oxidation pathway in the Y. lipolytica po1f. For this, we firstly constructed the engineering stains with the strengthened β-oxidation pathway by the combined overexpression of genes ylPOT1, ylMFE1, and ylPOX1-6. The experimental results showed that combined overexpression of genes ylPOT1, ylMFE1, and ylPOX4 or ylPOX5 significantly improve the cell growth of Y. lipolytica with using the edible oil as the substrate. Next, we introduced the Δ6-fatty acid desaturase encoding gene M-Δ6-D into the engineered strains with improved cell growth by the genomic integration method. As a result, we obtained a titer of 59.3 mg/L γ-linolenic acid from the engineered strain with using the gutter oil as substrate. The result set up a new stage of engineering Y. lipolytica as a sustainable biorefinery chassis strain for the synthesis of γ-linolenic acid with the gutter oil as substrate.
2 Engineering success
2.1 Constructing recombinant plasmids for overexpressing the β-oxidation pathway
2.1.1 PCR amplification of genes
Firstly, we amplified the sequences of genes ylPOT1, ylMFE1, ylPOX1, ylPOX2, ylPOX3, ylPOX4, ylPOX5 , and ylPOX6 with using the Y. lipolytica as the template by PCR method. The result of genes amplification has been showed in Figure 1.
Fig.1 Amplifying the fragments of genes ylPOX1 (a), ylPOX2 (b), ylPOX3 (c), ylPOX4 (d), ylPOX5 (e), ylPOX6 (f), ylPOT1 (g), and ylMFE1 (h)
2.1.2 Linearizing plasmid pYLXP’
The YaliBrick plasmid pYLXP’ was used as the expression vector in this project. Plasmid constructions were performed by using preciously reported methods (Lv et al., 2019). For linearizing plasmid, we used the nuclease SnaBI and KpnI to digest plasmid pYLXP’. The result of plasmid pYLXP’ digestion has been showed in Figure 2.
Fig.2 plasmid pYLXP’ digested by the nuclease SnaBI and KpnI
2.1.3 Construction of recombinant plasmids (the single-gene expression plasmids)
The recombinant plasmids for the single-gene expression (Table 1) were assembled by Gibson Assembly method with using linearized pYLXP’ (digested by SnaBI and KpnI) and the PCR-amplified fragments of genes, which were transformed into Escherichia coli DH5α. The selected marker is AMPr in E.coli, and the positive transformants were determined by colony PCR. The results of E. coli transformation plates and colony PCR have been showed in Figure 3 and Figure 4. The modified DNA fragments and plasmids were sequenced by Sangon Biotech (Shanghai, China).
Table 1 The single-gene expression plasmids
Fig.3 The plates of E. coli DH5α transformation
Fig.4 Colony PCR of the transformants. (a) The design of primers for colony PCR; (b) The results of colony PCR
2.1.4 Construction of recombinant plasmids (the multi-genes expression plasmids)
Multi-genes expression plasmids (Table 2) were constructed based on restriction enzyme subcloning with the isocaudamers AvrII and NheI. All genes were respectively expressed by the TEF promoter with intron sequence and XPR2 terminator. The selected marker is AMPr in E.coli. The results of transformation and colony PCR have been showed in Figure 5 and Figure 6.
Table 2 The multi-genes expression plasmids
Fig.5 The plates of E. coli DH5α transformation
Fig.6 Colony PCR of the transformants. (a) The design of primers for colony PCR; (b) The results of colony PCR
2.2 Genomic integration of the gene M-Δ6-D
2.2.1 PCR amplification of gene M-Δ6-D
Firstly, we amplified the fragment of gene M-Δ6-D (1365 bp) by PCR method. The template of gene M-Δ6-D was obtained from our lab. The result of genes amplification has been showed in Figure 7.
Fig.7 Amplifying the fragment of gene M-Δ6-D
2.2.2 Construction of the genomic integration plasmid
A marker-free gene knockout method based on Cre-lox recombination system was used as previously reported (Lv et al., 2019). The genomic integration plasmid were assembled by Gibson Assembly method with using linearized plasmid prDNAloxP (also be named as the plasmid P0, digested by AvrII and salI) and the gene M-Δ6-D fragment, and then transformed, into E. coli DH5α. The selected marker is AMPr in E.coli, and the positive transformants were determined by colony PCR. The modified DNA fragments and plasmids were sequenced by Sangon Biotech (Shanghai, China). The results of transformation and colony PCR have been showed in Figure 8 and Figure 9.
Fig.8 The plates of E. coli DH5α transformation
Fig.9 Colony PCR of the transformants
2.3 Testing the abilities of engineering yeasts for utilizing the edible oil
2.3.1 Yeast transformation
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 Figure10.
Fig.10 The plates of Y. lipolytica po1f transformation for overexpressing β-oxidation pathway
2.3.2 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 Figure11 and Figure 12. 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.11 Time profiles of cell growth of engineering strains
Fig.12 Seed culture and shaking flask cultivation of engineering strains
2.4 Producing γ-linolenic acid with using the edible oil by engineering yeasts
2.4.1 Genomic integration of gene M-Δ6-D into the improved strains
The procedure of genomic integration of gene M-Δ6-D into the improved strains (engineering strains po1f-pYLXP’-ylPOT1-ylMFE1-ylPOX4 and po1f-pYLXP’-ylPOT1-ylMFE1-ylPOX5) was described above, 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 13 and Figure14.
Fig.13 The plates of Y. lipolytica po1f transformation for introducing gene M-Δ6-D
Fig.14 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
2.4.2 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 15 and Figure 16. 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. 15 Gas chromatogram analysis of γ-linolenic acid titer in engineering strains
Fig. 16 Time profiles of engineering strains to produce γ-linolenic acid
3 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.
References
Lv, Y., Edwards, H., Zhou, J., Xu, P. 2019. Combining 26s rDNA and the Cre-loxP system for iterative gene integration and efficient marker curation in Yarrowia lipolytica. ACS Synth Biol.