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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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Growth characteristics, lipid content and CLA production of Y. lipolytica Polh-1292-spopai-d12-16 in batch fermentations with YNBD-SO medium. The best-performing strain, Polh-1292-spopai-d12-16, was cultivated in YNBD-SO medium for 168 hours under fermentation conditions. (A) The growth of Y. lipolytica was represented by lipid-free biomass calculated after subtraction of the cellular lipids from the total biomass. The glucose concentration was quantified using glucose oxidase in a standard glucose assay kit. (B), (C) Time course of lipid and CLA contents in cells and culture medium. Lipid and CLA yields in cells are expressed as percentage of total DCW. CLA titre in cells and medium and lipid titres in medium are expressed as g/l. The values represent the mean ± SD of three replicates.
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Figure 3: Growth characteristics, lipid content and CLA production of Y. lipolytica Polh-1292-spopai-d12-16 in batch fermentations with YNBD-SO medium. The best-performing strain, Polh-1292-spopai-d12-16, was cultivated in YNBD-SO medium for 168 hours under fermentation conditions. (A) The growth of Y. lipolytica was represented by lipid-free biomass calculated after subtraction of the cellular lipids from the total biomass. The glucose concentration was quantified using glucose oxidase in a standard glucose assay kit. (B), (C) Time course of lipid and CLA contents in cells and culture medium. Lipid and CLA yields in cells are expressed as percentage of total DCW. CLA titre in cells and medium and lipid titres in medium are expressed as g/l. The values represent the mean ± SD of three replicates.

Mentions: As shown in Figure 3, the cells were in a rapid growth phase from 0 to 38.5 h, with lipid-free biomass concentration increasing from 0.1 g/l to 17.1 g/l (Figure 3A). During that time, glucose was consumed rapidly and depleted by 24 h. Afterwards, the culture was in stationary phase from 38.5 h to 120 h, followed by a decline phase until the end of cultivation (168 h). Both biomass and growth medium were extracted for lipid analysis. Lipid content of the growth medium decreased from 18.7 g/l (the added soybean oil) to zero during the first 60 h of cultivation, indicating complete consumption of the soybean oil (Figure 3C). Meanwhile, total lipid and CLA in cells increased and reached peaks of 35% and 16% (w/w) of DCW, respectively, at 34 h (Figure 3B). The maximum CLA titre in cells was 3.1 g/l both at 34 h and 38.5 h (Figure 3B). Interestingly, CLA was also detected in the growth medium (Figure 3C). During the rapid growth phase, CLA content in growth medium increased continuously and the maximum CLA titre of 0.9 g/l was obtained at 38.5 h. After this time, CLA in cells and growth medium decreased sharply until no more CLA was detected at 168 h and 60 h, respectively.


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)

Growth characteristics, lipid content and CLA production of Y. lipolytica Polh-1292-spopai-d12-16 in batch fermentations with YNBD-SO medium. The best-performing strain, Polh-1292-spopai-d12-16, was cultivated in YNBD-SO medium for 168 hours under fermentation conditions. (A) The growth of Y. lipolytica was represented by lipid-free biomass calculated after subtraction of the cellular lipids from the total biomass. The glucose concentration was quantified using glucose oxidase in a standard glucose assay kit. (B), (C) Time course of lipid and CLA contents in cells and culture medium. Lipid and CLA yields in cells are expressed as percentage of total DCW. CLA titre in cells and medium and lipid titres in medium are expressed as g/l. The values represent the mean ± SD of three replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Growth characteristics, lipid content and CLA production of Y. lipolytica Polh-1292-spopai-d12-16 in batch fermentations with YNBD-SO medium. The best-performing strain, Polh-1292-spopai-d12-16, was cultivated in YNBD-SO medium for 168 hours under fermentation conditions. (A) The growth of Y. lipolytica was represented by lipid-free biomass calculated after subtraction of the cellular lipids from the total biomass. The glucose concentration was quantified using glucose oxidase in a standard glucose assay kit. (B), (C) Time course of lipid and CLA contents in cells and culture medium. Lipid and CLA yields in cells are expressed as percentage of total DCW. CLA titre in cells and medium and lipid titres in medium are expressed as g/l. The values represent the mean ± SD of three replicates.
Mentions: As shown in Figure 3, the cells were in a rapid growth phase from 0 to 38.5 h, with lipid-free biomass concentration increasing from 0.1 g/l to 17.1 g/l (Figure 3A). During that time, glucose was consumed rapidly and depleted by 24 h. Afterwards, the culture was in stationary phase from 38.5 h to 120 h, followed by a decline phase until the end of cultivation (168 h). Both biomass and growth medium were extracted for lipid analysis. Lipid content of the growth medium decreased from 18.7 g/l (the added soybean oil) to zero during the first 60 h of cultivation, indicating complete consumption of the soybean oil (Figure 3C). Meanwhile, total lipid and CLA in cells increased and reached peaks of 35% and 16% (w/w) of DCW, respectively, at 34 h (Figure 3B). The maximum CLA titre in cells was 3.1 g/l both at 34 h and 38.5 h (Figure 3B). Interestingly, CLA was also detected in the growth medium (Figure 3C). During the rapid growth phase, CLA content in growth medium increased continuously and the maximum CLA titre of 0.9 g/l was obtained at 38.5 h. After this time, CLA in cells and growth medium decreased sharply until no more CLA was detected at 168 h and 60 h, respectively.

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