Part:BBa_K3909022

pYLXP-ylPOT1-ylMFE1-ylPOX5

This composite part consist of pYLXP’-ylPOT1 (BBa_K3909010), pYLXP’-ylMFE1 (BBa_K3909011) and pYLXP’-ylPOX5 (BBa_K3909016). As shown in figure 1, our biobricks design is mainly divided into two parts: fatty acid degradation and γ-linolenic acid synthesis. The BBa_K3909022 is belong to part one -- fatty acid degradation.

Fig.1 The overview of our biobricks design.

We plan to enhance the oli degradation pathway by expressing three endogenous fatty acid degradation genes ylMEF1 (BBa_K3909006), ylPOT1 (BBa_K3909007), and ylPOXn (from BBa_K3909000 to BBa_K3909005), which are related to the metabolim of transforming acyl-CoA into acetyl-CoA in peroxisome (β-oxidation). Specifically, the β-oxidation includes three steps: i) oxidation, that catalyzed by six acyl-CoA oxidases (translated from ylPOX1 to ylPOX6); ii) hydration and dehydration, that catalyzed by multifunctional enzyme (translated from ylMFE1); and iii) thiolysis, that catalyzed by 3-ketoacyl-CoA thiolase (translated from ylPOT1)[1]. Firstly,we constructed the single gene-overexpressed plasmids by the aforementioned method, including pYLXP’-ylPOT1(BBa_K3909010), pYLXP’-ylMEF1 (BBa_K3909011) and pYLXP’-ylPOXn (from BBa_K3909012 to BBa_K3909017) , which are shown in Figure 2.

Fig.2 Single fragment plasmid construction.

For overpressing the whole oil degradation pathway, we further combined these genes by BioBrick method, and constructed plasmids pYXLP’-ylPOT1-ylMFE1-ylPOXn (from BBa_K3909018 to BBa_K3909023), as shown in figure 3. The Y. lipolytica (Po1f) we used cannot synthesize leucine by itself, so the Leu2 marker on the plasmid can be used as marker gene. Then, the constructed plasmids were transformed into Y. lipolytica Po1f.

Fig.3 Multi-fragment plasmid construction.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1343
  Illegal BamHI site found at 8015
  Illegal XhoI site found at 164
  Illegal XhoI site found at 565
  Illegal XhoI site found at 2378
  Illegal XhoI site found at 2840
  Illegal XhoI site found at 4249
  Illegal XhoI site found at 4341
  Illegal XhoI site found at 5161
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 832
  Illegal NgoMIV site found at 925
  Illegal NgoMIV site found at 1159
  Illegal AgeI site found at 1657
  Illegal AgeI site found at 1741
  Illegal AgeI site found at 1893
  Illegal AgeI site found at 2791
  Illegal AgeI site found at 4410
  Illegal AgeI site found at 5115
  Illegal AgeI site found at 5642
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2806
  Illegal BsaI site found at 4746
  Illegal BsaI site found at 5301
  Illegal BsaI site found at 6870
  Illegal BsaI site found at 7008
  Illegal BsaI.rc site found at 2
  Illegal BsaI.rc site found at 844
  Illegal BsaI.rc site found at 2216
  Illegal BsaI.rc site found at 4723
  Illegal BsaI.rc site found at 5400
  Illegal BsaI.rc site found at 5965
  Illegal BsaI.rc site found at 7192
  Illegal SapI site found at 4346
  Illegal SapI site found at 5501
  Illegal SapI.rc site found at 8130
  

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 4.

Fig.4 Amplifying the fragments of genes ylPOX1 (a), ylPOX2 (b), ylPOX3 (c), ylPOX4 (d), ylPOX5 (e), ylPOX6 (f), ylPOT1 (g), and ylMFE1 (h).

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[2]. 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 5.

Fig.5 plasmid pYLXP’ digested by the nuclease SnaBI and KpnI.

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 6 and Figure 7. The modified DNA fragments and plasmids were sequenced by Sangon Biotech (Shanghai, China).

Table 1 The single-gene expression plasmids.

Fig.6 The plates of E. coli DH5α transformation.

Fig.7 Colony PCR of the transformants. (a) The design of primers for colony PCR; (b) The results of colony PCR.

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 8 and Figure 9.

Table 2 The multi-genes expression plasmids.

Fig.8 The plates of E. coli DH5α transformation.

Fig.9 Colony PCR of the transformants. (a) The design of primers for colony PCR; (b) The results of colony PCR.

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 oC 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.10The plates of Y. lipolytica po1f transformation for overexpressing β-oxidation pathway.

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 oC 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 oC 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.

References：

[1] Green A , Silver P , Collins J , et al. Toehold switches: de-novo-designed regulators of gene expression.[J]. Cell, 2014, 159(4):925-939.

[2] 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.