Part:BBa_K3909024

M-Δ6-D

It is a Δ6-fatty acid desaturase gene from Mortierella alpina.

As shown in Figure 1, our biobricks design is mainly divided into two parts: fatty acid degradation and γ-linolenic acid synthesis. The BBa_K3909024 is belong to part two -- γ-linolenic acid synthesis.

Fig.1 The overview of our biobricks design.

Our goal is to synthesize γ-linolenic acid (GLA) on large scale. In order to convert α-linolenic acid into GLA[1], we are going to introduce Δ6-fatty acid desaturase gene from Mortierella alpina (M. alpina) into Y. lipolytica genome[2][3].

Y. lipolytica can produce linolenic acid, however, it lacks Δ6-fatty acid desaturase, which can catalyze the conversion of linolenic acid to γ-linolenic acid. Thus, we assembled Δ6-fatty acid desaturase gene M-Δ6-D (BBa_K3909024) into the hp4d-based expression vector p0, at the XPR2 terminator locus, to generate the recombinant vector p0-6-D. And to construct the complete γ-linolenic acid synthetic pathway, we integrated the correct vector into Y. lipolytica Po1f genome, yielding γ-linolenic acid producing strain.

Fig.2 the Δ6-fatty acid desaturase expression unit.

As shown in Figure 2, the Δ6-fatty acid desaturase expression unit is made up of five basic parts: Ampicillin resistance gene, URA3 selection marker, hp4d promoter, M-Δ6-D gene, and XPR2 terminator. Except for M-Δ6-D gene, which was intercepted and imported from M. alpina, the other four parts are all endogenous genes. Since UAR3 can encode lactoside 5-phosphate decarboxylase, which can catalyze one of the key reactions in the synthesis of yeast RNA pyrimidine nucleotides, it can be used as a selection marker. After being transformed into the protoplast of Y. lipolytica, this cassette can work to produce Δ6-fatty acid desaturase and transform oleic acid into the product γ-linolenic acid.

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 1259
  Illegal XhoI site found at 1087
  Illegal XhoI site found at 1246
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 535
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 361
  Illegal BsaI site found at 1206
  

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 3.

Fig.3 Amplifying the fragment of gene M-Δ6-D.

2. Construction of the genomic integration plasmid

A marker-free gene knockout method based on Cre-lox recombination system was used as previously reported[4]. 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 4 and Figure 5.

Fig.4 The plates of E. coli DH5α transformation.

Fig.5 Colony PCR of the transformants.

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 in part pages of BBa_K3909021 and BBa_K3909022. 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 6 and Figure 7.

Fig.6 The plates of Y. lipolytica po1f transformation for introducing gene M-Δ6-D.

Fig.7 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. 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 8 and Figure 9. 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. 8 Gas chromatogram analysis of γ-linolenic acid titer in engineering strains.

Fig.9 Time profiles of engineering strains to produce γ-linolenic acid.

C.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：

[1]B. Zhang, C. Rong, et al. “De novo synthesis of trans-10, cis-12 conjugated linoleic acid in oleaginous yeast Yarrowia lipolytica” Microb. Cell. Fact. 11 (2012) 51.

[2]Lauren T., Hal S. “ Production of α-linolenic acid in Yarrowia lipolytica using low-temperature fermentation” Applied Microbiology and Biotechnology 102(2018): 8809-8816.

[3]Meili Sun, Catherine M., et al. “Engineering Yarrowia lipolytica for efficient γ-linolenic acid production” Biochemical Engineering Journal 117(2017): 172-180.

[4]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.