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Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid.

Zhang B, Chen H, Li M, Gu Z, Song Y, Ratledge C, Chen YQ, Zhang H, Chen W - Microb. Cell Fact. (2013)

Bottom Line: In addition, trans-10, cis-12-CLA was also detected in the growth medium up to 0.9 g/l.We have successfully produced trans-10, cis-12-CLA with a titre of 4 g/l of culture (3.1 g/l in cells and 0.9 g/l in culture medium).Our results demonstrate the potential use of Y. lipolytica as a promising microbial cell factory for trans-10, cis-12-CLA production.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Conjugated linoleic acid (CLA) has been extensively studied for decades because of its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. We previously described the production of trans-10, cis-12 CLA in Yarrowia lipolytica by expressing the gene coding for linoleic acid isomerase from Propionibacterium acnes (pai). However the stable strain produced CLA at about 0.08% of dry cell weight (DCW), a level of production which was not high enough for practical applications. The goal of the present study was to enhance production of CLA by genetic engineering of Y. lipolytica strains.

Results: We have now co-expressed the delta 12-desaturase gene (FADS12, d12) from Mortierella alpina together with the codon-optimized linoleic acid isomerase (opai) gene in Y. lipolytica, expressed under the control of promoter hp16d modified by fusing 12 copies of UAS1B to the original promoter hp4d. A multi-copy integration plasmid was used to further enhance the expression of both genes. Using glucose as the sole carbon source, the genetically-modified Y. lipolytica produced trans-10, cis-12-CLA at a level of up to 10% of total fatty acids and 0.4% of DCW. Furthermore, when the recombinant yeast was grown with soybean oil, trans-10, cis-12-CLA now accumulated at a level of up to 44% of total fatty acids, which represented 30% of DCW after 38.5 h of cultivation. In addition, trans-10, cis-12-CLA was also detected in the growth medium up to 0.9 g/l.

Conclusions: We have successfully produced trans-10, cis-12-CLA with a titre of 4 g/l of culture (3.1 g/l in cells and 0.9 g/l in culture medium). Our results demonstrate the potential use of Y. lipolytica as a promising microbial cell factory for trans-10, cis-12-CLA production.

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Cell morphology of Y. lipolytica Polh-1292-spopai-d12-16. Cells were harvested after 24 h of cultivation in YNBD-SO medium. Optical microscopy analysis was performed to determine the integrity of Y. lipolytica cells under an Olympus System Microscope Model BX51. The images are presented at a 400× magnification.
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Figure 4: Cell morphology of Y. lipolytica Polh-1292-spopai-d12-16. Cells were harvested after 24 h of cultivation in YNBD-SO medium. Optical microscopy analysis was performed to determine the integrity of Y. lipolytica cells under an Olympus System Microscope Model BX51. The images are presented at a 400× magnification.

Mentions: To determine if the extracellular CLA was due to leakage from the cells, cell integrity of the best-performing strain in YNBD-SO medium was determined using microscopy. As Figure 4 shows, no obvious cell lysis had occurred, and a mixture of yeast-like and short mycelial cells were present at 24 h of fermentation in YNBD-SO.


Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid.

Zhang B, Chen H, Li M, Gu Z, Song Y, Ratledge C, Chen YQ, Zhang H, Chen W - Microb. Cell Fact. (2013)

Cell morphology of Y. lipolytica Polh-1292-spopai-d12-16. Cells were harvested after 24 h of cultivation in YNBD-SO medium. Optical microscopy analysis was performed to determine the integrity of Y. lipolytica cells under an Olympus System Microscope Model BX51. The images are presented at a 400× magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750285&req=5

Figure 4: Cell morphology of Y. lipolytica Polh-1292-spopai-d12-16. Cells were harvested after 24 h of cultivation in YNBD-SO medium. Optical microscopy analysis was performed to determine the integrity of Y. lipolytica cells under an Olympus System Microscope Model BX51. The images are presented at a 400× magnification.
Mentions: To determine if the extracellular CLA was due to leakage from the cells, cell integrity of the best-performing strain in YNBD-SO medium was determined using microscopy. As Figure 4 shows, no obvious cell lysis had occurred, and a mixture of yeast-like and short mycelial cells were present at 24 h of fermentation in YNBD-SO.

Bottom Line: In addition, trans-10, cis-12-CLA was also detected in the growth medium up to 0.9 g/l.We have successfully produced trans-10, cis-12-CLA with a titre of 4 g/l of culture (3.1 g/l in cells and 0.9 g/l in culture medium).Our results demonstrate the potential use of Y. lipolytica as a promising microbial cell factory for trans-10, cis-12-CLA production.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Conjugated linoleic acid (CLA) has been extensively studied for decades because of its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. We previously described the production of trans-10, cis-12 CLA in Yarrowia lipolytica by expressing the gene coding for linoleic acid isomerase from Propionibacterium acnes (pai). However the stable strain produced CLA at about 0.08% of dry cell weight (DCW), a level of production which was not high enough for practical applications. The goal of the present study was to enhance production of CLA by genetic engineering of Y. lipolytica strains.

Results: We have now co-expressed the delta 12-desaturase gene (FADS12, d12) from Mortierella alpina together with the codon-optimized linoleic acid isomerase (opai) gene in Y. lipolytica, expressed under the control of promoter hp16d modified by fusing 12 copies of UAS1B to the original promoter hp4d. A multi-copy integration plasmid was used to further enhance the expression of both genes. Using glucose as the sole carbon source, the genetically-modified Y. lipolytica produced trans-10, cis-12-CLA at a level of up to 10% of total fatty acids and 0.4% of DCW. Furthermore, when the recombinant yeast was grown with soybean oil, trans-10, cis-12-CLA now accumulated at a level of up to 44% of total fatty acids, which represented 30% of DCW after 38.5 h of cultivation. In addition, trans-10, cis-12-CLA was also detected in the growth medium up to 0.9 g/l.

Conclusions: We have successfully produced trans-10, cis-12-CLA with a titre of 4 g/l of culture (3.1 g/l in cells and 0.9 g/l in culture medium). Our results demonstrate the potential use of Y. lipolytica as a promising microbial cell factory for trans-10, cis-12-CLA production.

Show MeSH
Related in: MedlinePlus