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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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Relative copy numbers of integrated expression cassettes in Y. lipolytica multi-copy transformants. Real-time PCR was used to estimate the copy numbers of the integrated expression cassettes among 12 Polh-1292-spopai-d12 transformants. Y. lipolytica Polg was used as a control organism with a single copy of both the ura3 and suc2 target sequences. As ura3, opai and d12 coexisted in expression cassettes, the copy numbers of three genes were considered to be equal. Error bars represent standard deviations from biological triplicates.
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Figure 1: Relative copy numbers of integrated expression cassettes in Y. lipolytica multi-copy transformants. Real-time PCR was used to estimate the copy numbers of the integrated expression cassettes among 12 Polh-1292-spopai-d12 transformants. Y. lipolytica Polg was used as a control organism with a single copy of both the ura3 and suc2 target sequences. As ura3, opai and d12 coexisted in expression cassettes, the copy numbers of three genes were considered to be equal. Error bars represent standard deviations from biological triplicates.

Mentions: Twelve selected transformants with the multi-copy integration cassette derived from plasmid pINA1292-spopai-d12 were investigated. The copy numbers of the co-expression cassettes were estimated using the data obtained by real-time PCR analysis. Y. lipolytica Polg was used as a control organism with a single copy of both ura3 and suc2 target sequences. As ura3, opai and d12 coexisted in expression cassettes, the copy numbers of three genes were considered to be equal. Distribution of opai / d12 copy numbers analyzed in twelve isolated transformants is shown in Figure 1. For all the clones tested, copy numbers fell in a narrow range of four to eight copies, with an average of five to six copies/cell. The strain with the highest copy number was Polh-pINA1292-spopai-d12-16 (8 copies).


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)

Relative copy numbers of integrated expression cassettes in Y. lipolytica multi-copy transformants. Real-time PCR was used to estimate the copy numbers of the integrated expression cassettes among 12 Polh-1292-spopai-d12 transformants. Y. lipolytica Polg was used as a control organism with a single copy of both the ura3 and suc2 target sequences. As ura3, opai and d12 coexisted in expression cassettes, the copy numbers of three genes were considered to be equal. Error bars represent standard deviations from biological triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Relative copy numbers of integrated expression cassettes in Y. lipolytica multi-copy transformants. Real-time PCR was used to estimate the copy numbers of the integrated expression cassettes among 12 Polh-1292-spopai-d12 transformants. Y. lipolytica Polg was used as a control organism with a single copy of both the ura3 and suc2 target sequences. As ura3, opai and d12 coexisted in expression cassettes, the copy numbers of three genes were considered to be equal. Error bars represent standard deviations from biological triplicates.
Mentions: Twelve selected transformants with the multi-copy integration cassette derived from plasmid pINA1292-spopai-d12 were investigated. The copy numbers of the co-expression cassettes were estimated using the data obtained by real-time PCR analysis. Y. lipolytica Polg was used as a control organism with a single copy of both ura3 and suc2 target sequences. As ura3, opai and d12 coexisted in expression cassettes, the copy numbers of three genes were considered to be equal. Distribution of opai / d12 copy numbers analyzed in twelve isolated transformants is shown in Figure 1. For all the clones tested, copy numbers fell in a narrow range of four to eight copies, with an average of five to six copies/cell. The strain with the highest copy number was Polh-pINA1292-spopai-d12-16 (8 copies).

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