Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid
- Baixi Zhang†1,
- Haiqin Chen†2,
- Min Li1, 2,
- Zhennan Gu2,
- Yuanda Song2,
- Colin Ratledge3,
- Yong Q Chen1,
- Hao Zhang2Email author and
- Wei Chen1Email author
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 5 February 2013
Accepted: 22 May 2013
Published: 16 July 2013
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.
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.
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.
Conjugated linoleic acids (CLA) are geometric and positional dienoic isomers of linoleic acid (18:2, n-9). Over the last three decades, CLA has been extensively studied because of its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and immune system modulation . The multiple beneficial physiological effects of CLA can be attributed to the two most studied isomers, with cis-9, trans-11 CLA playing the most important role in the anticancer effects, and trans-10, cis-12 CLA having a predominant effect on weight management and prevention of atherogenesis [2, 3]. For the purpose of research and applications in medicinal and nutritional fields, each CLA isomer needs to be available at high purity and in large quantity. However, the chemically-produced CLA is a mixture of four (8,10-, 9,11-, 10,12-, and 11,13-18:2) cis/trans positional isomers . As a result, biological processes to produce single CLA isomers have been investigated [5–10]. To date, the highest yield of cis-9, trans-11 CLA (10.5 mg/ml) was obtained in Delacroixia coronata using trans-vaccenic acid methyl ester as substrate . By contrast, the bioprocess yield of the other isomer, trans-10, cis-12 CLA was very low, with a mere 0.2 mg/ml in recombinant Escherichia coli using exogenous linoleic acid (LA) as substrate .
In our previous study , we constructed a de novo trans-10, cis-12 CLA biosynthesis system by transforming the oleaginous yeast, Yarrowia lipolytica, with the recombinant linoleate isomerase gene (pai) from Propionibacterium acnes. Codon usage optimization of pai and multi-copy integration significantly improved the expression of the linoleate isomerase gene in Y. lipolytica. The amount of trans-10, cis-12 CLA reached 5.9% of total fatty acids, and 0.23% of DCW . However, the yeast strain with the highest integrated copy number (24 copies) was unstable and the yield of CLA gradually decreased to 0.08% of DCW. This yield therefore was not high enough for further development. In an attempt to further increase trans-10, cis-12 CLA production, we genetically modified Y. lipolytica to improve the expression of linoleate isomerase gene, and modified fermentation conditions to increase the concentration of the isomerase substrate, LA. In this manner, the yield of trans-10, cis-12 CLA was improved significantly.
Construction of recombinant yeast strains
Polh-pINA1312-opai, the single-copy integration strain with the codon-optimized opai gene described in our previous study , was used as a control and as a model strain to develop CLA high-producing recombinant strains. To increase LA content in Y. lipolytica, the delta12-desaturase gene (d12) was cloned from M. alpina to construct opai -d12 co-expression cassettes. Delta12-desaturase converted oleic acid (18:1) to LA (18:2). Polh-pINA1312-opai-d12 was obtained by transforming Polh with opai-d12 co-expression cassettes. To enhance opai expression, the promoter, hp4d, was replaced by a modified promoter, hp16d, located upstream of opai, resulting in the recombinant strains Polh-pINA1312-spopai and Polh-pINA1312-spopai-d12. Furthermore, the spopai -d12 co-expression cassettes were also placed in a multi-copy integration plasmid, pINA1292. The new multi-copy integration cassette was transformed to obtain the strain Polh-pINA1292-spopai-d12. Eight to twelve transformants in each series were picked randomly and confirmed to be all positive by PCR analysis.
opai / d12 copy numbers in Polh-1292-spopai-d12 transformants
Effect of genetic modifications on opai transcript and protein levels
Effect of genetic modifications on CLA production
Fatty acid composition and accumulation in cellular lipids of Y. lipolytica strains, cultivated on YPD medium
Polh-1312-spo pai- d12-9
Polh-1292-spo pai- d12-7
Polh-1292-spo pai- d12-16
8.2 ± 0.4
11.2 ± 0.3
9.5 ± 0.6
11.8 ± 0.9
13.9 ± 0.7
13.3 ± 0.5
11.3 ± 0.7
6.4 ± 0.8
11.3 ± 1.2
10.5 ± 0.7
12.4 ± 0.8
10.7 ± 1.1
2.6 ± 0.3
1.0 ± 0.0
0.7 ± 0.1
0.4 ± 0.0
2.2 ± 0.2
2.3 ± 0.1
2.1 ± 0.1
2.4 ± 0.2
2.5 ± 0.2
2.4 ± 0.3
0.3 ± 0.0
1.3 ± 0.1
1.2 ± 0.0
1.3 ± 0.2
0.6 ± 0.0
1.1 ± 0.1
49.7 ± 2.0
13.2 ± 0.5
48.8 ± 1.6
25.9 ± 1.2
21.9 ± 0.8
26.4 ± 1.0
27.8 ± 1.1
62.2 ± 2.2
26.0 ± 1.3
44.8 ± 1.7
40.3 ± 0.9
35.9 ± 0.5
10 t, 12c-CLA
0.4 ± 0.1
0.7 ± 0.1
1.1 ± 0.2
2.2 ± 0.4
7.8 ± 0.7
9.8 ± 0.5
TFA (% of DCW)
4.2 ± 0.2
4.0 ± 0.1
4.8 ± 0.4
4.5 ± 0.2
3.7 ± 0.1
3.9 ± 0.3
Fatty acid analysis (Table 1) showed that in all strains containing recombinant delta12-desaturase, a large proportion of oleic acid (OA) had been converted to LA, which was now the major fatty acid. In addition, delta9,12-16:2 was detected as a new fatty acid in opai-d12 co-expressing strains, suggesting that the delta12-desaturase can also use 16:1 as substrate .
Transformants are stable
The stability of the recombinant Y. lipolytica strains was tested under nonselective conditions by culturing Polh-pINA1292-spopai-d12-16 in YPD for 2 weeks (approximately 160 generations). No change was detected in the copy numbers of opai, lipid content and fatty acid compositions of Polh-pINA1292-spopai-d12-16 obtained in YPD (data not shown), indicating that the best-performing transformant was stable even after 160 generations.
Production of CLA by fermentation in YNBD-SO media
Fatty acid composition in cellular and extracellular lipids of Polh-1292-sp opai-d12-16 in fermentations with YNBD-SO medium
Polh-1292-spo pai- d12-16
Cell - 0 h
Cell - 38.5 h
Medium - 0 h
Medium – 38.5 h
12.2 ± 0.6
9.3 ± 0.4
9.9 ± 0.5
11.8 ± 0.6
7.9 ± 0.8
0.9 ± 0.1
1.7 ± 0.2
0.3 ± 0.0
0.9 ± 0.1
3.6 ± 0.2
3.5 ± 0.1
6.3 ± 0.3
15.7 ± 1.0
20.2 ± 0.7
19.4 ± 0.9
23.0 ± 1.2
60.6 ± 2.6
14.6 ± 0.6
57.5 ± 2.2
22.5 ± 0.7
3.4 ± 0.1
8.0 ± 0.5
2.1 ± 0.3
10 t, 12c-CLA
0.3 ± 0.0
44.3 ± 2.1
30.4 ± 1.1
The fatty acid compositions of cells and culture medium at 0 and 38.5 h of cultivation are shown in Table 2. The LA contents in cells and growth medium decreased from 61% to 15% and from 58% to 23%, respectively. In contrast, the CLA contents in cells and growth medium increased from 0.3% to 44% and zero to 30% respectively, suggesting that LA in the soybean oil added to the medium was efficiently converted to CLA by PAI. In addition, lipid analysis of culture medium at 38.5 h of cultivation revealed that extracellular CLA existed exclusively in free fatty acid form. At the same time point, about 90% of the intracellular CLA existed in free fatty acid form. Total CLA production in the fermenter using YNBD-SO was almost 4 g/l, about 90 times higher compared to that using YPD medium in shaking flask cultures (45 mg/l, Figure 2).
PAI activity in culture supernatant
To determine whether the CLA detected in culture medium was due to PAI secretion, LA isomerase activity was tested in the growth medium. Yeast cultures were harvested after 24 h by centrifugation. One ml of cell-free medium supplemented with 400 μg free LA (as the substrate for PAI ) was incubated at 37°C for 1 h, and then extracted for lipid analysis. The data showed that no LA had been converted to CLA. This suggested PAI secretion had not occurred.
Cell integrity of Y. lipolytica in YNBD-SO medium
In our previous study, we constructed a de novo CLA biosynthesis system by transforming the oleaginous yeast, Y. lipolytica, with the recombinant linoleate isomerase gene from P. acnes. The expression level of opai was increased by the optimization of the codon-usage of pai and multi-copy integration of the gene. The average copy numbers of opai in pINA1292-opai transformants were 12–13 copies/cell, with a maximum of 24 copies/cell, in a strain which was unstable after a few passages of cultivation, and the copy number decreased to 7 copies/cell.
In this study, the average copy number of opai integrated in cells using a multi-copy integration vector was estimated at 5–6 copies/cell. The lower number of integrated copies obtained in the present work might result from the much longer integrated fragments (7827 bp) than in previous study (4311 bp). In this study, the best-performing strain, Polh-pINA1292-spopai-d12-16, which contained 8 copies of the expression cassette, remained stable in copy numbers and CLA yields (9.8% of total fatty acids). This is consistent with a previous study showing that an average of 10 copies/cell was stable in the Polh-pINA1292 expression system . In addition to multi-copy integration, enhanced function of the promoter can also increase the expression level of opai, which appears to be one of the bottlenecks for CLA production in recombinant Y. lipolytica.
Another potential bottleneck for CLA production in this system is the intracellular concentration of the substrate, LA. Our study showed that co-expression of d12 with opai increased the LA content in cells from 28% to 62% of the total fatty acids; however, the CLA content increased only slightly (from 0.4% to 0.7%) (Table 1). A possible explanation is that the LA being produced by the delta12-desaturase is in an esterified form, which is not a substrate for PAI. An in vitro study has demonstrated that PAI, unlike fatty acyl desaturases and fatty acyl elongases, uses free fatty acid as its only substrate . However, the accumulation of esterified LA may contribute to a slightly increased intermediate pool of free LA and indirectly increase CLA production.
Because PAI is a soluble protein in the cytoplasm, it may compete for the free LA with other cytosolic enzymes that convert free LA to esterified forms of LA (methyl-, CoA-, etc.) in various pathways including oxidation, desaturation, elongation or re-esterification . Competition for the same substrate by the enzymes involved in these pathways may then limit the flux of free LA towards CLA production. Hence, the overall efficiency of the CLA synthesis depends on the expression level of PAI and free LA content in Y. lipolytica.
In addition, a decrease in LA content (from 62% to 45%) in the opai-d12 co-expression strain was found when the hp4d promoter located upstream of opai was replaced by the hp16d enhanced promoter. The hp4d promoter comports 4 copies of the UAS1B enhancer site upstream of the core promoter, whereas hp16d has 16 UAS1B copies. This decrease in LA is probably due to promoter interference between hp16d driving the opai and the weaker hp4d promoter driving the d12-desaturase gene.
Due to its ability to hydrolyze extracellular lipids, Y. lipolytica has been efficiently cultivated on various hydrophobic substrates such as alkenes or lipids to produce many extra- or intra-cellular metabolites of industrial significance . Indeed, in this study, soybean oil, which is rich in LA, was immediately hydrolyzed and absorbed by Y. lipolytica and free LA was efficiently converted to CLA. This suggests that exogenous LA is a good substrate for PAI, unlike de novo synthesized LA. Additionally, about a quarter of the total CLA production was recovered in the growth medium. As no PAI extracellular activity could be detected in the culture supernatant, these results suggest that CLA present in the medium was not a result of secreted PAI. Furthermore, no cell fragmentation was observed, indicating that CLA was not liberated into the medium by cell lysis. In addition, lipid analysis revealed that the extracellular CLA was in the free fatty acid form.
A possible mechanism that may account for the appearance of CLA in the growth medium is that hydrolysis of the oil, due to the very high activity of lipases in this yeast, may produce free fatty acids faster than they can be incorporated into triacylglycerols and other intracellular lipids. Consequently, as the cell does not accumulate free fatty acids, the latter have to be pumped out of the cells and thus appear in the culture medium as free fatty acids, which include the newly-synthesized CLA. As all the enzymes involved in fatty acid metabolism, including the linoleate isomerase, are able to act on the fatty acids in exogenous soybean oil, CLA production is significantly increased. If Y. lipolytica could be coaxed into continuously secreting CLA, this transformed yeast strain would be an efficient cell factory able to convert low-cost plant oils into valuable CLA which can be directly collected from the culture medium without biomass extraction processes.
We constructed a Y. lipolytica strain able to produce large quantities of trans-10, cis-12-CLA. This was achieved by combining different genetic engineering approaches to enhance PAI expression and to increase substrate availability.
When the final transformant was cultivated in medium supplemented with soybean oil, 3.1 g CLA/l (16% of DCW) was produced in cells with an additional 0.9 g CLA/l in the growth medium. This result is the highest production of trans-10, cis-12-CLA to date and shows promise for its large-scale production.
Strains and culture conditions
The auxotrophic strain Y. lipolytica Polh  was used as the PAI expression host, while Y. lipolytica Polg was used as a control strain for real-time PCR. Y. lipolytica strains were kindly provided along with the plasmids pINA1312 and pINA1292  by Prof. Catherine Madzak (Institute National de la Recherche Agronomique/AgroParisTech, France). Escherichia coli DH5α was used for routine sub cloning and plasmid propagation.
Media and growth conditions for E. coli were described by Sambrook et al. , and those for Y. lipolytica were described by Barth and Gaillardin . YPD and YNBD media were prepared for Y. lipolytica as described previously . YPD contained 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) dextrose. YNBD contained 0.17% (w/v) yeast nitrogen base (without amino acids and ammonium sulphate), 0.5% (w/v) ammonium sulphate and 2% (w/v) dextrose. YNBD-SO medium used for fermentation is YNBD with 0.15% yeast extract, 0.5% (w/v) NH4Cl, 0.01% (w/v) uracil, 2% (w/v) Casamino acids and 2% (w/v) soybean oil. 2% (w/v) soybean oil in 0.02% (w/v) Tween 80 was prepared as a 10-X sonicated stock emulsion and sterilized by filtration through a 0.22 μm membrane .
Typically, cultivation was performed as follows. From YNBD plates, Y. lipolytica transformants were grown in YNBD (10 ml in 50 ml Erlenmeyer flasks, 200 rpm, 28°C, 48 h). These pre-cultures were inoculated into YPD (50 ml in 250 ml Erlenmeyer flasks) to an OD600 of 0.1 and further cultured for another 72 h at 28°C and 200 rpm. Cells were collected by centrifugation (6000 g, 5 min) and washed once with de-ionized water, and the pellets were either directly used for real-time PCR and western blot analysis or lyophilized for biomass determination and lipid analysis.
For fermentation experiments, a seed culture was grown in YNBD (150 ml in 500 ml Erlenmeyer flasks, 200 rpm, 28°C, 48 h). The 150 ml seed culture was used to inoculate 3 l YNBD-SO containing 2% (w/v) soybean oil in a fermenter (5 l, BioFlo Celligen 115, New Brunswick). The fermentation was run at 28°C for 7 days. The aeration was at 3 l/min and the stirrer was set at 500 rpm. The pH was maintained at 6.0 with either 3 M KOH or 2 M H2SO4. Silicone oil was periodically added as an antifoam agent. Ten ml samples were taken at intervals. Cells were harvested by centrifugation (8000 g, 15 min) and washed twice with 0.85% (w/v) NaCl and the pellets were lyophilized for biomass determination and lipid analysis. The lipid-free biomass was calculated after subtraction of the cellular lipids from the total biomass. The cell-free culture was used for the LA isomerase activity assay, and heated at 80°C for 10 min (to inactivate lipases) for the glucose concentration assay and lipid analysis.
Plasmids construction and transformation
Standard protocols were followed for DNA manipulation . Vector pINA1312 and pINA1292 both contain hp4d promoter which comprised four tandem UAS1B copies upstream from a minimal leucine promoter. Previously, UAS1B was found to be an enhancer element and hybrid promoters containing multiple UAS1B sequences successfully amplified the expression levels of hrGFP and lacZ[20, 21]. In this study, the hp16d promoter was constructed by fusing 12 tandem UAS1B enhancer sequences to the hp4d promoter. The four tandem UAS1B sequence fragment, UAS1B4 was synthesized (Genscript, Nanjing, China), flanked with Cla I-Hin dIII-Spe I sites on the 5′-end and Mlu I-Xba I sites on the 3′-end. The synthetic UAS1B4 sequence was inserted into pUC57 to form pUC-UAS1B4. The UAS1B4 fragment was extracted from pUC-UAS1B4 by digestion with Hin dIII and Xba I restriction enzymes and ligated with pUC-UAS1B4 cut with Hin dIII and Spe I, resulting pUC-UAS1B8. The construction of pUC-UAS1B12 was performed following the same procedures used in the case of pUC-UAS1B8. The UAS1B12 fragment was extracted from pUC-UAS1B12 by digestion with Cla I and Mlu I restriction enzymes and inserted into pINA1312 and pINA1292 to form pINA1312sp and pINA1292sp, respectively. The codon-optimized LA isomerase sequence (opai)  was digested with Pml I and Kpn I and then inserted into pINA1312sp and pINA1292sp to form pINA1312-spoPAI and pINA1292-spoPAI. The delta 12-desaturase sequence (FADS12, d12) was isolated from Mortierella alpina ATCC 32222 cDNA by PCR as previously described . The primers for delta12-desaturase amplification were d12f: (5′-CCGCACGTGATGGCACCTCCCAACA-3′) and d12r: (5′-GGGGTACCTTACTTCTTGAAAAAGACCACG-3′). After amplification, the PCR product was digested with Pml I and Kpn I and ligated with pINA1312 to form pINA1312-d12. The d12 expression cassette fragment containing the hp4d promoter, flanked with Eco RI, was amplified using pINA1312-d12 as template, with primers d12Expf (5′-GGAATTCTATCGATACGCGTGCATGCTGAG-3′) and d12Expr (5′-CATGGGACACGGGCATCTC-3′). The d12 expression cassette fragment was digested with Eco RI and ligated with pINA1312-opai, pINA1312-spopai and pINA1292-spopai, resulting in the expression vectors pINA1312-opai-d12, pINA1312-spopai-d12 and pINA1292-spopai-d12, respectively. All newly constructed plasmids were screened by restriction enzyme digestion and PCR, and then confirmed by DNA sequencing.
Four new expression cassettes from the plasmids constructed above were transformed into Y. lipolytica strain Polh, resulting in four recombinant strains, Polh-pINA1312-opai-d12, Polh-pINA1312-spopai, Polh-pINA1312-spopai-d12 and Polh-pINA1292-spopai-d12. Transformation was performed by the lithium acetate method, as described previously . Transformants were selected by plating on YNBD.
Real-time PCR and western blotting
Real-time PCR was used to determine the transcript level of opai and estimate the copy number of the expression cassettes. The DNA, RNA extraction and real-time PCR were performed as described elsewhere [23, 24]. The primer sequences encoding the opai were: opaiF (5′-TCCCGATTACGCTGACAAGAC-3′) and opaiR (5′-CTCCATGTCATCCAGCACCAT-3′). Western blot analysis was used to determine the expression level of PAI protein and carried out according to methods described previously .
Lipid extraction and fatty acid analysis
The cells, grown in YPD medium, were collected by centrifugation (6000 g, 5 min) and washed once with deionised water, and the pellet was lyophilized prior to lipid extraction. The cells, grown in YNBD-SO medium, were collected by centrifugation (8000 g, 15 min), washed twice with 0.85% w/v NaCl-0.5% bovine serum albumin solution and the pellet was lyophilized. The cell-free culture and washing solution were combined for lipid extraction. Lipids from the equivalent weight of freeze-dried cells (20 mg) were directly transmethylated with 10% (v/v) methanolic HCl at 50°C for 3 h . The lipids in medium were extracted twice with a mixture of chloroform/methanol (1:1) and total fatty acids in the medium were similarly methylated with 10% (v/v) methanolic HCl. Free fatty acids in cells and medium were methylated with 20 μL (trimethylsilyl)-diazomethane and 400 μL methanol at 37°C for 30 min. Fatty acid methyl esters (FAMEs) were analyzed by GC .
Stability of the transformants
Strains were grown in YPD for 2 weeks to keep the cells in the exponential phase of growth throughout the experiment. Cells were cultured in 20 ml of medium in 100-ml shaking flasks. Every morning, fresh cultures were inoculated at an initial A600 of 0.5 and grown for 8 h. In the evening, fresh cultures were inoculated at an initial A600 of 0.2 and grown for 16 h. Samples of the previous culture were used to inoculate the new culture. Under our growth conditions, one cell generation corresponded to 2 h.
Determination of glucose concentration
Glucose was quantified by using glucose oxidase in a standard glucose assay kit (RSbio, Shanghai. China).
Extracellular enzymatic activity assay
To determine extracellular LA isomerase activity in YNBD-SO medium, 1 ml cell-free culture, with or without 400 μg free LA, was incubated for 1 h at 37°C. Fatty acids from the reaction system were extracted, methylated and analyzed via GC as described above.
This work was supported by the National Science Fund for Distinguished Young Scholars (31125021), the National Natural Science Foundation of China (No. 21276108, 31271812, 31171636), the National High Technology Research and Development Program of China (2011AA100905, 2012AA022105C), the National Basic Research Program of China 973 Program (2012CB720802), the 111 project B07029, and the Fundamental Research Funds for the Central Universities (No. JUSRP51320B).
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