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Research Paper
Co-expression of heterologous desaturase genes in Yarrowia lipolytica Lu-Te Chuang1, Dzi-Chi Chen2, Jean-Marc Nicaud3, Catherine Madzak3, Ying-Hsuan Chen2 and Yung-Sheng Huang4 1
Department of Biotechnology, Yuanpei University, Hsin Chu, Taiwan Yeastern Biotech Co., Ltd., Taipei, Taiwan 3 UMR1238 Microbiologie et Ge´ne´tique Mole´culaire, INRA-CNRS-AgroParisTech, Thiverval-Grignon 78850, France 4 Department of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan 2
The hybrid promoter (hp4d) expression cassette, one of the efficient tools of Yarrowia lipolytica expression system, has been applied to produce or secrete a variety of recombinant proteins. This cassette directs a strong gene expression, because the hp4d promoter exhibits high level quasi-constitutive activity. The objective of this study is to test whether two expression cassettes inserted into a vector could function efficiently and simultaneously. Taking advantage of the well-known biosynthesis pathway of glinolenic acid (GLA), we examined the performance of Y. lipolytica, transformed with two expression cassettes containing previously cloned D12-desaturase and D6-desaturase genes, by monitoring fatty acid composition of cellular lipids. Our results confirmed that each individual desaturase gene was expressed efficiently by the expression cassette. When two cassettes with respective desaturase genes, carried on the same vector, were integrated into yeast genome, a significant level of GLA was synthesized from endogenous linoleic acid (LA) and oleic acid (OA). Besides, both expression cassettes functioned effectively without influence from each other. These findings indicated that co-expression of two desaturase genes by this dual cassette vector was effective and simultaneous. Results from the present study provide an alternative approach for both the production of several proteins at the same time, and the development of single cell oil containing high-valued polyunsaturated fatty acids (PUFAs).
Introduction Heterologous gene expression is crucial for the production of proteins in industrial applications. For years, the well-established eukaryotic expression system, Saccharomyces cerevisiae, has been widely utilized to express genes and synthesize proteins in different aspects. However, certain limitations were found as industrial practicing with this system, such as poor plasmid stability, low product yield and difficulties in scaling-up production. In order to find alternative expression tools, a number of non-conventional yeasts, including Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris, and so on have been explored for many Corresponding author: Huang, Y.-S. (
[email protected]) 1871-6784/$ - see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2010.02.006
years [1]. Among these strains, Y. lipolytica is thought as a potential substitute: not only has this yeast species been applied in industries for production of organic acids and single cell proteins with GRAS (generally regarded as safe) status, but also it was one of the most attractive host organisms for heterologous production [2]. Besides, the genome of Y. lipolytica has been sequenced and is available on the Genolevures website (http://cbi.labri.fr/Genolevures/elt/YALI), and genetically modified strains and various expression vectors have been developed [3–6]. One series of expression vectors constructed by Madzak et al. contains an hp4d promoter, which is a strong hybrid promoter carrying four tandem copies of an upstream activator sequence (UAS1B) from the promoter of the XPR gene (pXPR2) fused to a minimal LEU promoter www.elsevier.com/locate/nbt
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(pLEU2) [3]. Unlike the frequently used Yarrowia promoter (pXPR2), this quasi-constitutive recombinant promoter directs protein expression constantly without multiple influences by nutritional and environmental factors in medium, such as carbon/nitrogen sources and pH values [3]. A plasmid carrying the expression cassette constituted by the hybrid promoter followed by a cloned gene of interest can be integrated into the genome of a genetically modified Y. lipolytica strain by homologous recombination. This efficient hp4d promoter expression cassette system has been applied to produce various proteins, such as b-galactosidase, prorennin [3], cytokinin oxidase [7], lipase [4], laccases [8,9], and so on. Since the hp4d containing expression cassette expresses a recombinant protein effectively, it is possible that two (or more) expression cassettes in a single vector could allow to express different proteins efficiently and independently. To test this hypothesis, we took advantage of the well-known pathway for g-linolenic acid (GLA; D6,9,12-18:3) production: oleic acid (OA; D9-18:1) is first D12-desaturated to form linoleic acid (LA; D9,12-18:2) and subsequently converted to GLA by the action of D6-desaturase. Therefore, in this study, based on the fact that Y. lipolytica contains certain levels of endogenous OA and LA [10], we inserted two previously cloned desaturase cDNAs (D12-desaturase and D6-desaturase) originating from Mortierella alpina [11] into two expression cassettes carried on a same plasmid, to examine whether they could be coexpressed, allowing the synthesis of higher levels of GLA.
Experimental procedures Chemicals, biological materials and culture media Gas chromatography standard (GLC-461) was obtained from NuChek Prep, Inc. (Elysian, MN, USA). Yeast extract, peptone and yeast nitrogen base were purchased from Difco Laboratories (Detroit, MI, USA). Triheptadecanoin (used as an internal standard; ISTD), ammonium sulfate and dextrose were from Sigma (St. Louis, MO, USA). Hexane was UV grade and other solvents were distilled-in-glass quality. Three types of culture media were used in this study. The YPD medium contained 1% of yeast extract, 2% of peptone and 2% of dextrose. The 1/20 YP medium was applied to cultivate transformed yeast, and per liter contained 0.5 g of yeast extract, 1 g of peptone and 3% of dextrose. For the purpose of selecting transformants, the YNBD plate, which contained 1.72 g of yeast nitrogen base, 5 g of ammonium sulfate, 20 g of dextrose and 2% of agar was prepared.
Plasmid construction, yeast transformation and gene expression The cDNA sequences corresponding to D6-desaturase (AF110510) and D12-desaturase (AF110509) from M. alpina were obtained as
previously described [11], and were inserted into pYLEX1 expression vector from YLEX kit. The Y. lipolytica Expression Kit (YLEX kit for expression of recombinant protein in Y. lipolytica) was purchased from Yeastern Biotech Co., Ltd. (Taipei, Taiwan). The name pINA1269 previously assigned to this pYLEX1 vector [3], was preferably used in this study. Briefly, a pair of primers [Delta 6AATG (F) and Delta 6-Xcml (R)] with homology to the sequences downstream of initiation site and upstream of stop codon of D6desaturase, respectively, was designed, according to the specifications of YLEX kit (Table 1). PCR amplification was run on a DNA thermal cycler (Applied Biosystem, Foster City, CA) following the program of 30 s at 94 8C, 30 s at 55 8C, and 1.5 min at 72 8C for 35 cycles, followed by extension for 5 min at 72 8C. After amplification, D6-desaturase PCR products were digested with XcmI restriction endonuclease and ligated to pINA1269 vector restricted with PmlI and XcmI, to construct the plasmid pYLd6 (Figure 1a). Similarly, the primer Delta12-AATG (F) and Delta12-BamHI (R) were used to amplify D12-desaturase cDNA (Table 1). To construct the vector expressing D12-desaturase gene (pYLd12), the PCR fragment was digested with BamHI restriction enzyme and ligated to pINA1269 vector restricted with PmlI and BamHI (Figure 1b). The newly constructed plasmids were screened by restriction enzyme digestion and PCR, and then confirmed by DNA sequencing. To construct two plasmids for simultaneous expression of D6-desaturase and D12-desaturase genes, we designed a pair of primers with homology to, respectively, the 50 -end and the 30 -end of the sequence of both D6-desaturase and D12-desaturase expression cassettes (hp4d promoter/D6-desaturase or D12-desaturase gene/ terminator) from either pYLd6 or pYLd12 plasmid. The forward primer Delta6-12F and the reverse primer Delta6-12R were used together with linear pYLd12 or pYLd6 for a PCR amplification (Table 1) as described above. The newly amplified blunt-ended PCR fragment isolated from pYLd12 and the plasmid pYLd6 digested with NruI restriction enzyme were ligated to form the co-expression plasmid pYLd12d6 (Figure 1c); and the other amplified blunt-ended PCR fragment, isolated from pYLd6, and the plasmid pYLd12 digested with NruI restriction enzyme were ligated to form the co-expression plasmid pYLd6d12 (Figure 1d). The control (empty pINA1269 vector) and the four newly constructed plasmids were linearized using NotI enzyme, and introduced into the genetically modified Y. lipolytica strain, Po1g (Leu, DAEP, DAXP, Suc+, pBR322 integration platform) [3]. Transformation of Y. lipolytica was performed using the one-step method previously described by one of us [12]. Transformants were selected by plating on YNBD plates (minimal medium without leucine), and screened by PCR on yeast colonies and lipid analysis.
TABLE 1
Primers used in this study Primers
Sequence 0
Description 0
Delta6-AATG (F) Delta6-XcmI (R)
5 Ph-AATGGCTGCTGCTCCCAGTG-3 50 -GCCAGAGTCGGACTGGTTACTGCGCCTTACCCATC-30
Amplification for D6-desaturase XcmI restriction site underlined
Delta12-AATG (F) Delta12-BamHI (R)
50 Ph-AATGGCACCTCCCAACACTATC-30 50 -GTGGATCCTTACTTCTTGAAAAAGACCACG-30
Amplification for D12-desaturase BamHI restriction site underlined
Delta6-12F Delta6-12R
50 -GGTCCGGACCCAGTAGTAGGTTGAGGC-30 50 -TTTCCGGACTTATCATCGATGATAAGCTCTC-30
Amplification for co-expression D6-desaturase and D12-desaturase
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FIGURE 1
Map of the plasmids used to transform Yarrowia lipolytica Po1g strain for expression of, respectively, Mortierella alpina D6-desaturase gene (a), D12-desaturase gene (b), and construction of co-expression plasmids (c and d).
To examine whether the two respective desaturase cDNAs could be expressed in the expression system in Y. lipolytica, randomly picked transformants Po1g(pYLd6) and Po1g(pYLd12) were first grown overnight in YPD medium at 28 8C, and these pre-cultures (1 108 cells) were used to inoculate 100 ml of 1/20 YP medium for 72 h. This cultivation medium was previously shown to allow stable expression of desaturase genes by Yarrowia cells (data not shown). In order to determine if both desaturase genes could be expressed efficiently and independently, selected transformants of the newly constructed Po1g(pYLd12d6) and Po1g(pYLd6d12) strains were cultivated in 1/20 YP medium at 28 8C for 72 h. In all studies, the expression of both desaturase cDNA was represented by the conversion of substrates to products (i.e. D6desaturation and D12-desaturation), and calculated by the ratio of [product]/([product] + [substrate]) 100%. The Po1g strain transformed with the empty vector [Po1g(pINA1269)] was used as the negative control.
Lipid extraction and fatty acid analysis After cultivation, cells were harvested by centrifugation, and the cell pellet was washed once with sterile deionized water. Total lipids from transformed yeasts were extracted following the procedure described previously [13]. Briefly, the rinsed cells were extracted with 30 ml of chloroform:methanol (2:1, v/v) at 4 8C overnight. The lipids in chloroform were separated from the aqueous phase by adding 6 ml of saline solution and collected, and the solvent was evaporated using a stream of nitrogen. The extracted total lipids were saponified and methylated to generate fatty acid methyl esters (FAME). FAME were then analyzed by gas chromatography (GC) using an Agilent 6890 gas chromatograph equipped with a flame-ionization detector and a fused-silica capillary column (Omegawax; 30 m 0.32 mm, i.d. film thickness 0.25 mm, Supelco, Bellefonte, PA, USA). Fatty acids were identified by comparing their retention times to those of known standards, and quantified using the internal standard. www.elsevier.com/locate/nbt
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Statistical analysis Data were analyzed by analysis of variance (ANOVA) and Fisher’s protected least significant difference (LSD) to compare differences between means of conversion rates. Means differences were considered significant at the P 0.05 level.
Results Conversion of LA to GLA by D6-desaturase and OA to LA by D12-desaturase Research Paper
Linoleic acid is the major polyunsaturated fatty acid synthesized by Y. lipolytica [10]. In order to convert the endogenous LA to GLA, the plasmid pYLd6 containing D6-desaturase gene from M. alpina was introduced into the genetically modified strain of Y. lipolytica, Po1g. The transformed yeast po1g(pYLd6) was then incubated in 1/20 YPD medium at 28 8C for 72 h. Results in Figure 2a show that a significant portion (60%) of the endogenous LA has been converted to GLA. There were also trace amounts of D6,9-16:2 (2.3%) and D6,9-18:2 (4.1%), the D6-desaturation products of palmitoleic acid (D9-16:1) and oleic acid (D9-18:1), respectively. None of these three D6-fatty acids was found in the control yeast transformed with empty vector [Po1g(pINA1269)] (Figure 2a). To determine whether D12-desaturase cDNA could also be expressed in Yarrowia expression system, the transformed yeast po1g(pYLd12) was incubated as above described. When fatty acid composition was analyzed by GC, it showed that a large proportion of OA (67%) in the transformed yeast was converted to LA, whereas only 32% of OA was converted to form LA in the control strain
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(Figure 2b). Results in Figure 2b also show the appearance of a small additional peak in the transformed yeasts [Po1g(pYLd12)], but not in the control yeasts. This minor novel fatty acid was tentatively identified as D9,12-16:2 as discussed previously [11]. This finding indicates an efficient conversion of the endogenous OA to LA by action of the inserted recombinant D12-desaturase gene.
Co-expression of fungal D6-desaturase and D12-desaturase genes in the transformed Y. lipolytica Results described above demonstrate that D6-desaturase or D12desaturase genes were significantly expressed in the transformed Y lipolytica. Next, we further examined if D12-desaturase and D6desaturase genes inserted into two expression cassettes could be coexpressed by measuring the level of GLA biosynthesized from the endogenous OA and LA. Results in GC chromatogram (Figure 2c and d) show a high peak of GLA appearing only in yeasts transformed with both desaturase genes but not in the controls. However, there were also trace amounts of four additional peaks in the chromatogram from Po1g(pYLd12d6) or Po1g(pYLd6d12). Three of them were identified as D6,9-16:2, D9,12-16:2 and D6,9-18:2 as previously described [11]. The fourth novel fatty acid was tentatively identified as D6,9,12-16:3, a possible D6-desaturation product of D9,12-16:2.
Comparison of gene expression (fatty acid composition) in five control or transformed Y. lipolytica strains Five different strains of control or transformed Y. lipolytica have been used in this study, and Table 1 shows the comparison of fatty
FIGURE 2
Gas chromatogram of fatty acids of total lipids in the yeasts transformed with empty vector [Po1g(pINA1269)] and the yeasts carrying the Mortierella D6-desaturase gene [Po1g(pYLd6)] (a), D12-desaturase gene [Po1g(pYLd12)] (b) or both D6-desaturase and D12-desaturase genes [Po1g(pYLd12d6)] (c) or [Po1g(pYLd6d12)] (d). Solid arrows show the presence of D6-desaturation or D12-desaturation products, including D6,9-16:2, D9,12-16:2, D6,9,12-16:3, D6,9-18:2 or g-linolenic acid (D6,9,12-18:3). Open arrow shows the presence of linoleic acid (D9,12-18:2), a fatty acid found in all the transformed yeasts. Heptadecanoic acid (17:0) in the chromatograms was used as the internal standard (ISTD). 280
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FIGURE 3
Rates of D12-desaturation and D6-desaturation exhibited by five strains of transformed Yarrowia lipolytica. D6-desaturation or D12-desaturation was calculated as [product]/[product + substrate] 100%. Each value point represents the mean of three independent experiments. In each category, values with different letters are significantly different from each other at P < 0.05.
acid composition in their lipids. When the fatty acid profile of Po1g(pYLd12) was analyzed, it showed that the percentage of LA in the cellular lipids in this new yeast strain was twofold higher than that in the control strain (39% versus 19%). The lipids extracted from Po1g(pYLd6), a yeast strain carrying the fungal D6-desaturase gene, contained a higher percentage of OA compared to the control strain (53% versus 41%), while it converted most of its LA to GLA. When both D12-desaturase and D6-desaturase genes were co-expressed, levels of LA and GLA in both po1g(pYLd12d6) (12% and 20%) and po1g(pYLd6d12) (16% and 20%) increased nearly fourfold compared to those when only D6-desaturase gene was expressed (3% and 5%, respectively). The level of OA, on the other contrary was decreased by around twofold (53% in Po1g(pYLd6) versus 26% in Po1g(pYLd12d6) or 24% in po1g(pYLd6d12)). We have also calculated the rates of conversion from OA to LA (D12-desaturation) and from LA to GLA (D6desaturation) in these yeast strains. Results in Figure 3a show that Po1g(pYLd12) strain displayed the highest rate of converting OA to LA (67%), followed by Po1g(pYLd6d12) (60%), Po1g(pYLd12d6) (55%), Po1g(pINA1269) (32%) and Po1g(pYLd6) (13%). However, the rates of D6-desaturation (conversion of LA to GLA) among the three D6-desaturase-transformants were comparable (Figure 3b).
Discussion The hp4d promoter expression cassette in an expression/secretion vector has been applied to produce or secrete significant levels of various recombinant proteins in previous reports (reviewed in Ref. [5]). We constructed a novel plasmid with two cassettes in order to co-express two heterologous genes simultaneously. Our results demonstrated that this dual expression cassette vector drove a strong expression of two fungal desaturase genes. When the plasmids pYLd6 and pYLd12, containing each expression cassette with the respective desaturase gene, was introduced into the yeast, it exhibited high degrees of conversion of substrates to products (Figure 3), especially D6-desaturation of LA to GLA. Approximately 60% of LA was converted to GLA, which was significantly higher than rates of D6-desaturation reported previously [11,14,15]. Although rates of D6-desaturation were chan-
ged significantly when the transformed yeast Po1g(pYLd6) was cultivated in a variety of media and temperatures (data not shown), the D6-desaturase gene was consistently expressed without severe repression, a characteristics of hp4d promoter that makes it more versatile than other Yarrowia promoters requiring induction by nutrients or chemicals [5]. These findings are consistent with the previous observations that hp4d promoter exhibited a strong quasi-constitutive activity, and was not repressed by culture conditions such as preferred carbon or nitrogen sources, or acidic environment [3]. Results in this study also illuminated that both strains of transformed yeasts po1g(pYLd12d6) and Po1g(pYLd6d12) produced up to 20% of GLA from endogenous LA and OA (Table 2), and that the rates of D12-desaturation and D6-desaturation were also comparable, as high as 60% (Figure 3). It indicated that two expression cassettes, carried on a single vector, expressed both fungal desaturases simultaneously and efficiently. In addition, our results revealed that the respective position of the two cassettes in the vector (Figure 1c and d) does not seem to affect the expression of the two genes: similar results were obtained with Po1g(pYLd6d12) and Po1g(pYLd12d6). The other question related to the future applications of the dual expression vector is whether the two expression cassettes would negatively influence each other when the plasmid was introduced into yeasts. In Figure 3a, the expression of D6-desaturase gene into Y. lipolytica resulted in a significant decrease in the rate of D12-desaturation, as shown by the comparison between Po1g(pINA1269) and Po1g(pYLd6) (decrease from 32.5% to 17%). We cannot explain why this decrease occurred, but it is possible that expression of D6-desaturase driven by strong hp4d promoter repressed that of endogenous D12-desaturase. Decreasing effects were also found between Po1g(pYLd12) and Po1g(pYLd12d6) (or Po1g(pYLd6d12)) (Figure 3a), but to a lesser extent (from 66.6% to 55.4% or 56%, respectively). The reason accounting for a lesser degree of decrease between Po1g(pYLd12) and Po1g(pYLd12d6) (or Po1g(pYLd6d12)) was probably due to the fact that both dual cassette plasmids contained the expression cassette with fungal D12-desaturase gene. The efficient expression of D12-desaturase gene compensated partly www.elsevier.com/locate/nbt
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TABLE 2
Percentages of oleic acid (OA), linoleic acid (LA) and g-linolenic acid (GLA) in total lipids from five transformed strains of Y. lipolytica Transforming plasmid
pINA1269 (control) b
pYLd12
pYLd6 d
OA
40.7 1.5
19.4 1.9
LA
19.4 1.22
38.7 1.11
GLA
–
pYLd12d6
pYLd6d12
c
26.3 2.7
24.2 2.1c
3.3 0.15
12.5 0.44
15.8 1.13
x
y
20.2 1.1y
a
53.5 0.9
4.9 0.1
–
20.0 1.9
Values with different superscripts (letters or numbers) on the same roll are significantly different (P < 0.05).
Research Paper
the potential negative influence on endogenous D12-desaturase resulting from D6-desaturase expression. By contrast, no such effect was observed on the rate of D6-desaturation when D12-desaturase gene was introduced into Y. lipolytica (Figure 3b). Co-expression of genes of interest in yeast is usually achieved with two or more plasmids as previously described [11,16], however, we clearly showed that two expression cassettes inserted into pINA1269 vector were able to function effectively and independently. This new process might be applied for expressing two (or possibly more) different genes, or several copies of one gene, in a single transformation step. In addition, Y. lipolytica is able to produce and accumulate high levels of cell lipids [17,18]. Taking advantage of its oleaginous characteristic, we could possibly synthesize various polyunsaturated fatty acids by inserting more
expression cassettes with the corresponding genes such as elongase, D6-desaturase or D5-desaturase, and so on (carried on mono-copy vectors such as pINA1269, but using other selection markers), or changing for a more adequate multiple-copy vector carrying the corresponding genes (as exemplified in Ref. [4]). Such further studies would possibly lead to producing high-valued single cell oil. In conclusion, by determining fatty acid profile of cellular lipids, we confirmed that the hp4d promoter drove a strong expression of desaturase genes, and further showed that two expression cassettes could co-express heterologous genes efficiently and simultaneously. This design could be applied to the production of several proteins at the same time, or to the biosynthesis of high-valued PUFA oil through molecular engineering.
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