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Microbiology & Fermentation Technology

Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates

Jiancai Wang Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, China; School of Life Sciences, University of Science and Technology of China, Hefei, China, Ronghua Xu College of Life Sciences, Anhui Science and Technology University, Fengyang, China, Ruling Wang Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, China, Mohammad Enamul Haque Key Laboratory for Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China & Aizhong Liu Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, China; Key Laboratory for Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, ChinaCorrespondenceliuaizhong@mail.kib.ac.cn
Pages 1214-1222
Received 12 Nov 2015
Accepted 23 Dec 2015
Published online: 11 Feb 2016
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Microbiology & Fermentation Technology

Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates

Abstract

The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

Graphical abstract

Scheme of metabolic pathways and enzymes of S. cerevisiae.

Key words: Lipomyces starkeyi, acetyl-CoA carboxylase, glycerol 3-phosphate dehydrogenase, fatty acid biosynthesis, Saccharomyces cerevisiae

Rising crude oil prices, unsustainable supply, and concerns over climate change have become increasingly important to search for renewable substitutes. Biodiesel is one of such alternative fuels, but using plant oils for producing biodiesel is a costly and unsustainable path.1) Microbial oils, as feedstock for producing biodiesel, have great advantages over that of plant oils, such as adaptability to diverse feedstocks, reduced land requirements, short life cycle, and easier scale-up.2) Using microbial oils as feedstock for producing biodiesel has attracted great interests in industry.

Some oleaginous yeast species, such as Lipomyces sp., Rhodosporidium sp., and Yarrowia sp., can accumulate intracellular lipids as high as 50% of their dry cell weight (DCW).3) In particular, Lipomyces starkeyi, originally isolated from soil, accumulates lipids up to 60% of its dry weight and the fatty acid composition highly similar to vegetable oils.2,4,5) Furthermore, L. starkeyi is capable of utilizing some low-cost raw materials to produce microbial oils, such as sewage sludge, fishmeal wastewater, sunflower meal hydrolysate, potato starch wastewater, and willow wood sawdust hydrolysate.4,6–9) Also, the complete genome information of L. starkeyi has been released and available to the public (http://genome.jgi.doe.gov/Lipst1_1/Lipst1_1.home.html), and the gene transformation systems for L. starkeyi have been recently established.10,11) These studies provide great opportunities to further engineer its genetic background for producing targeted strains for scale-up in industry. Although microbial oleaginity is known for decades, knowledge on the mechanisms controlling lipid accumulation in oleaginous yeasts remains limited. Hence, there is an immediate need for investigating the genetic mechanisms controlling lipid accumulation in oleaginous yeasts, specifically, identifying the key rate-limiting enzymes responsible for lipid biosynthesis and characterizing the functions of genes encoding these rate-limiting enzymes.

The biosynthesis process of microbial oils (in the form of triacylglycerols, TAGs) involves in de novo fatty acid synthesis and TAG assembly through Kennedy pathway at the endoplasmic reticulum and lipid bodies.12) The TAG assembly consumes acyl-CoAs using substrate glycerol 3-phosphate (G3P) with diverse enzymes. There, G3P is formed from sugars by the reduction of dihydroxyacetone phosphate (DHAP) concomitant with NADH oxidation catalyzed by cytosolic glycerol 3-phosphate dehydrogenase (GPD) (E.C.1.1.1.8).13) The subsequent dephosphorylation of G3P leads to the formation of glycerol by glycerol 3-phosphatase (GPP) (E.C. 3.1.3.21) (Fig. 1).14)

Fig. 1. Scheme of metabolic pathways and enzymes of S. cerevisiae.

Abbreviations: DHAP, dihydroxyacetone phosphate; G3P, glycerol 3-phosphate; TAG, triacylglycerol; ACC1, acetyl-CoA carboxylase; FAS1, FAS2, fatty acid synthase; GPD1/2, glycerol 3-phosphate dehydrogenase; GPP1/2, glycerol 3-phosphatase; GUT2, mitochondrial FAD-dependent glycerol 3-phosphate dehydrogenase.
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The process of de novo fatty acid synthesis produces acyl-CoAs in cytosol. Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA which is the first committed step in fatty acyl-CoAs biosynthesis and has been postulated to be a rate-limiting step in the process of cytosolic TAG synthesis, as shown in Fig. 1. Previous studies have described the successful genetic improvement in lipid accumulation by modifying the activity of ACC.15–18) The genes coding for the ACC enzyme (EC 6.4.1.2) have been authenticated from several organisms.19,20) In comparison with prokaryotes and plastids of most plants, mammals, fungi, and yeasts contain multifunctional ACC enzymes, which possess three functional components biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT) in a single polypeptide. Furthermore, there are two isoforms of the ACC: cytosolic and mitochondrial enzymes.21,22) However, little is known about the function of ACC in regulating lipid biosynthesis from oleaginous and non-oleaginous yeasts.

In this study, we cloned the full-length L. starkeyi acetyl-CoA carboxylase gene (LsACC1) from oleaginous L. starkeyi and characterized its sequence structure. In particular, we performed the functional analysis of LsACC1 by transferring it into non-oleaginous Saccharomyces cerevisiae with the overexpression of the endogenous S. cerevisiae glycerol 3-phosphate dehydrogenase gene (GPD1). Also, we compared the functions of LsACC1 and the endogenous S. cerevisiae acetyl-CoA carboxylase gene (ScACC1) in S. cerevisiae. The current study provides new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

Materials and methods

Organisms, growth conditions, and plasmids

L. starkeyi AS 2.1560 obtained from the China General Microbiological Culture Collection Center (CGMCC) was used as a genetic resource and was maintained as described previously.3) It was maintained at 4 °C on YPD agar slants containing 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, and 1.5% (w/v) agar and subcultured twice a month. The nutrient-rich medium used for the preculture was YPD medium. The nitrogen-deficient medium used for lipid production contained: KH2PO4 (12.5 g/L), Na2HPO4 (1 g/L), (NH4)2SO4 (0.1 g/L), MgSO4·7H2O (2.5 g/L), CaCl2·2H2O (0.25 g/L), yeast extract (0.9 g/L), glucose (36 g/L), and 0.625 mL trace element solution. The trace element solution contained (g/L): FeSO4·7H2O 16, MnSO4·H2O 4, Al2(SO4)3·18H2O 5.52, CoCl2·6H2O 2.92, ZnSO4·7H2O 0.8, Na2MoO4·2H2O 0.8, CuCl2·2H2O 0.4, H3BO3 0.2 and KI 1.6 in 5 N hydrochloric acid. The S. cerevisiae YA103 (MATa gpp1::kanMX4 gpp2::HIS3)23) was used as the recipient for expression of the cloned gene LsACC1. The yeast was grown in YPD medium at 30 °C. Escherichia coli Trans1-T1 (TransGen, Beijing, China) was used for plasmid propagation and was grown at 37 °C in a Luria–Bertani medium (LB) supplemented with 100 μg/mL of ampicillin when necessary. The pX2-GAL vector contains the ampicillin resistance gene, GAL1 promoter for inducing expression, and the LEU2 selectable marker. The pWXY1.0 contains the ampicillin resistance gene, GAL1 promoter for inducing expression, and the TRP1 selectable marker.24) The two vectors were used for gene expression in yeast. The pEASY™-T5 Zero Cloning Vector (TransGen, Beijing, China) was used for DNA sequencing. All media were autoclaved at 121 °C for 15 min.

Genetics techniques

All basic DNA manipulation procedures were performed according to Sambrook and Russell.25) Primers used in this study synthesized by Generay Corporation (Shanghai, China) are listed in Table 1. All sequencing was performed at the Beijing Genome Institute (Shenzhen, China), and all plasmids were prepared by High-purify Plasmid DNA Mini-preparation Kit (Bioteke, China).

Table 1. Primers used in this study.

Culture conditions of L. starkeyi

Preculture of L. starkeyi AS 2.1560 was made in YPD medium at 30 °C for 72 h. Lipid production was performed in 250-mL Erlenmeyer flasks containing 50 mL of the nitrogen-deficient medium. The cultures were initiated with OD600 = 0.4 and incubated in a rotary shaker at 200 rpm, 30 °C. Samples (2 mL each) were withdrawn at 12 h (sample “12-h”), 48 h (sample “48-h”) and 96 h (sample “96-h”). The collected samples were flash frozen in liquid nitrogen and stored at −80 °C until total RNA extraction. Parallel samples were also collected for biomass and lipid content determination as described below.

DNA extraction

L. starkeyi AS 2.1560 was grown in 50 mL YPD media at 30 °C on a rotary shaker until OD600 reached 5.0. A sample (2 mL) of cells was harvested by centrifugation (5000 × g, 5 min) and was suspended in 500 μL extraction buffer (2% Triton X-100, 100 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA, 1% SDS, 0.1% β-mercaptoethanol, pH 8.0). Lysis of yeast cells was achieved by addition of acid-washed 0.4–0.6 mm diameter glass beads, and continuous vortexing for 10 min at the highest speed utilizing a vortex MM400 (Retsch, Germany). The suspension of disrupted cells was purified by adding equal volumes of phenol–chloroform–isoamylalcohol (25:24:1), followed by isopropanol precipitation. Subsequently, the pellet was washed by 70% ethanol and dissolved in 50 μL TE containing 50 μg/mL RNaseA. The DNA concentration was measured using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Cloning of the LsACC1 gene and sequencing

The part sequences of 5′ and 3′ terminal of the LsACC1 gene were amplified by PCR with two pairs of degenerated primers (LsACC1-F1, LsACC1-R1, and LsACC1-F2, LsACC1-R2 in Table 1), which were designed based on conserved amino acid sequences WAGWGHA, QFGHIFA and SFGPQED, DQMEMYA, respectively. After getting the partial sequence of LsACC1, the unknown sequences between 5′ and 3′ terminal were amplified by the primers LsACC1-F3 and LsACC1-R3. To clone the full-length LsACC1 gene, the other unknown flanking sequences were amplified by TAIL-PCR.26) The specific primers for the 5′ and 3′ terminal sequences were LsACC1, LsACC2, LsACC3 and 3SP1, 3SP2, 3SP3, respectively. The arbitrary degenerate (AD) primers were AD1, AD2, AD3, AD4, AD5, and AD6 (Table 1), and the PCR cycle conditions were the same as those described by Liu and Chen.26) Amplified flanking sequences were cloned into pEASYTM-T5 Zero Cloning Vector and validated by sequencing. The full-length sequence of LsACC1 was obtained through all assembly sequences. The sequence of the LsACC1 gene has been deposited in the GenBank database under the accession no. KJ948118.

Sequence analyses and real-time quantitative PCR (qPCR)

Nucleotide sequences and amino acid sequences were primarily analyzed using Vector NTI advance 11.0 software (Invitrogen, USA). Multiple sequence alignment and construction of the phylogenetic tree were performed using the neighbor-joining criteria in MEGA5 with 1000 bootstrap replicates.27)

The collected samples of 12 h, 48 h, and 96 h were disrupted with glass beads, and total RNA was extracted following the standard protocol of TRIZOL reagent (Invitrogen, USA). The RNA samples were quantified by NanoDrop 2000 spectrophotometer. QPCR was performed to validate the transcription levels of LsACC1. Total RNA (1 μg) was reverse transcribed into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China). Relative expression levels of LsACC1 were quantified using SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, Dalian, China) with a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). L. starkeyi actin gene (LsACT1) as the endogenous reference gene was used to normalize the amount of the total mRNA in all samples.

Plasmid construction and genes expression in yeast

To express the GPD1, the open reading frame (ORF) of GPD1 was amplified from S. cerevisiae genomic DNA using primers GPD1-1 and GPD1-2 (Table 1). The GPD1 was ligated to the SmaI site of pX2-GAL to yield pX2-GPD1 by homologous recombination in E. coli Trans1-T1.28) To express the LsACC1 and ScACC1, the coding sequences (CDSs) of the genes were amplified from the cDNA of L. starkeyi AS 2.1560 and S. cerevisiae genomic DNA using primers LsACC1-3, LsACC1-4 and ACC1-1, ACC1-2, respectively (Table 1). The LsACC1 and ScACC1 were ligated to the XhoI/KpnI site of pWXY1.0 to yield pLSA1.0 and pSCA1.0 by homologous recombination in E. coli Trans1-T1, respectively.28)

The plasmid was transformed into YA103 via the lithium acetate method as described previously.29) The transformed cells were selected on SC-L, SC-T, or SC-TL agar plates (0.67% bacto-yeast nitrogen base without amino acids, 2% glucose and a mixture of appropriate nucleotide bases and amino acids without leucine, tryptophan or tryptophan and leucine, 2% agar) at 30 °C for 3–4 days. Expression of recombinant genes was induced by cultivation of the transformants in nitrogen-limited SD-L, SD-T, or SD-TL medium containing 0.17% bacto-yeast nitrogen base without amino acids and ammonium sulfate, 2% galactose and a mixture of appropriate nucleotide bases and amino acids without leucine, tryptophan or tryptophan and leucine, 1 g/L ammonium sulfate.30) To test whether overexpression of the GPD1 gene can accumulate lipid in yeast, the transgenic yeasts with pX2-GAL or pX2-GPD1 were fed on oleic acid (C18:1), respectively. Oleic acid was added into the SD-L media to a final concentration 2 mg/mL with 2 mg/mL Tween-20 for making oleic acid dissolve completely. The cultures were grown to the stationary phase at 30 °C.

Determination of intracellular G3P in S. cerevisiae

To extract the intracellular G3P from S. cerevisiae, an aliquot of cultures containing approximately 1.5 × 109 cells was harvested by centrifugation (5000 × g for 5 min) and was suspended in 1 mL phosphate buffer (pH 7.0). Lysis of yeast cells was achieved by addition of acid-washed 0.4–0.6 mm diameter glass beads, and continuous vortexing for 10 min at the highest speed utilizing a vortex MM400. The resulting suspension was cleared by centrifugation at 13000 × g for 10 min, and the G3P was measured using an ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. The samples were separated on a Waters Acquity UPLC type BEH C18 (100 mm × 2.1 mm, 1.7 μm) analytical column with a mobile phase of A (methanol) and B (5 mM formamide solution) (v/v = 10:90). The flow rate was set at 0.2 mL/min, and the injection volume was 1 μL. For operation in the MS/MS mode, the electrospray ion source was operated with negative ion modes in a single run. The ESI parameters were set as follows: source temperature, 150 °C; desolvation temperature, 250 °C; desolvation gas flow, 10 L/h. The optimized cone voltages were 20 V. The multiple reaction monitoring mode using specific precursor/product ion transitions was employed for quantification. The molecular ions of G3P were fragmented at collision energy of 15 eV using argon as collision gas. Ion detection was performed by monitoring the transition: m/z 170.95 → 96.96 → 78.97.

The standards G3P (35%, w/w, AKSci, USA) were dissolved in methanol to make stock solutions at a concentration of 437.5, 43.75, and 4.375 μg/mL, respectively. The working solutions were diluted by methanol to construct calibration curves at the designated concentration ranges, and then, the samples were analyzed by UPLC-MS/MS.

Determination of lipid content

Total lipids of cultured cells were extracted by the method of Zhao et al. 3) Briefly, yeast cells were harvested from 20 mL culture broth and dried at 105 °C to constant weight to give the DCW (g/L). In parallel, yeast cells harvested from 20 mL culture medium were digested with 4 M HCl at 78 °C for 1 h, and then, the total lipids were extracted using the chloroform/methanol (1:1, v/v) method. The extracts were washed with 0.1% NaCl and distilled water, dried over anhydrous Na2SO4, and evaporated in vacuo, and the residue was dried at 105 °C overnight to give the total cellular lipid. The lipid content (%) was expressed as gram lipid per gram DCW.

RNA extraction and qPCR analysis

S. cerevisiae YA103 cells of stationary phase were harvested by centrifugation, disrupted with glass beads, and RNA was extracted by the method described in section “Sequence analyses and real-time quantitative PCR (qPCR)”. QPCR was performed to validate the transcription levels of GPD1 and ScACC1, respectively. Total RNA (1 μg) was reverse transcribed into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time). Relative expression levels of GPD1 and ScACC1 were quantified using SYBR® Premix Ex Taq™ (Tli RNaseH Plus) with a CFX96 Real-Time PCR Detection System, respectively. ACT1 as the endogenous reference gene was used to normalize the amount of the total mRNA in all samples.

Results and discussion

Identification of the LsACC1 gene

The partial LsACC1 gene was cloned using degenerate PCR amplification, and we got two fragments about 1094 and 1264 bp. The full-length sequence of LsACC1 gene (7786 bp), including the complete ORF (from position 207 to 7138 bp, total 6932 bp) was obtained using TAIL-PCR approach as described in method section.

Multiple alignment analysis showed that amino acids sequences of LsACC1 exhibited a high homology BC, BCCP, and CT domains with other acetyl-CoA carboxylase gene (ACC) from several organisms tested, particularly in regions of the conserved amino acid sequences for the ATP-, biotin-, carboxybiotin-, and acetyl-CoA-binding sites (Fig. 2(A)). The CDS of LsACC1 gene encoding a protein of 2234 amino acids spanned 6705 bp with a calculated molecular mass of 248 kDa and an isoelectric point (pI) of 6.078. The subsequent sequence analysis showed an ORF composed of 6932 bp containing four putative introns. These introns were 66, 57, 56, and 48 bp in length and were located at nucleotides +124 to +189, +2833 to +2889, +2972 to +3027, and +4522 to +4569, respectively, relative to the initiation codon (ATG). In the NCBI database, amino acid alignment of L. starkeyi acetyl-CoA carboxylase protein (LsACC1) with other proteins presented that it had high homology with other yeasts ACC proteins (ACCs) that correspond to the result of phylogenetic analysis (Fig. 2(B)). The amino acid sequence identities of the LsACC1 to the ACCs of yeasts, animals, and cytosol of plants are 65–70, 47, and 41–45%, respectively.

Fig. 2. The structural organization of the conserved domains in ACC and phylogenetic relationships of multi-domain ACC proteins (ACCs) from various organisms.

Notes: (A) The deduced amino acid sequence of the ATP-, biotin-, carboxybiotin-, and acetyl-CoA-binding sites of LsACC1 (L. starkeyi acetyl-CoA carboxylase) was aligned with those from BfACC1 (Botryotinia fuckeliana acetyl-CoA carboxylase), GlACC1 (Glarea lozoyensis acetyl-CoA carboxylase), AfACC1 (Aspergillus fumigatus acetyl-CoA carboxylase), and ScACC1 (S. cerevisiae acetyl-CoA carboxylase) in box. Identical amino acid residues are shaded in black, while conserved residues are shaded in gray. (B) The plant, animal, and yeast ACCs are in the same branch, respectively. AtACC1, Arabidopsis thaliana NP_001185143; AtACC2, A. thaliana NP_174850; DmACC1, Drosophila melanogaster NP_001097277; DrACC1, Danio rerio XP_005165611; DrACC2, D. rerio XP_005165252; HsACC1, Homo sapiens NP_942131; HsACC2, H. sapiens O00763; LsACC1, L. starkeyi KJ948118; MmACC1, Mus musculus Q5SWU9; MmACC2, M. musculus NP_598665.2; PpACC1, Physcomitrella patens XP_001754424; PpACC2, P. patens XP_001773073; RnACC1, Rattus norvegicus NP_579938; RnACC2, R. norvegicus CBU93243; SbACC1, Sorghum bicolor XP_002446178; SbACC2, S. bicolor XP_002442242; ScACC1, Saccharomyces cerevisiae NP_014413; ScHFA1, S. cerevisiae NP_013934; XtACC1, Xenopus tropicalis NP_001131086; XtACC2, X. tropicalis XP_004911956; YlACC1, Yarrowia lipolytica XP_501721.
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Three conserved catalytic domains BC, BCCP, and CT existing in the LsACC1 were found using the NCBI conserved domain search31) and similar to the previous reports for ACC from other eukaryotic organisms, such as animals and fungi in addition to ACC in the cytosol of plants.32) Moreover, a common sequential arrangement of the domains, NH2-BC-BCCP-CT-COOH, was also found in the LsACC1, in which the BC (residues 39–550) and BCCP (residues 614–749) domains were located at the N-terminus, whereas the CT domain was located in the C-terminal sequence from residues 1573 to 2124 (Fig. 2(A)). Within the BC domain, a putative binding site for ATP (residues 226–245) was found. In addition, the two proline residues located 24 and 32 residues upstream from the putative biotin-binding site may be involved in the formation of a hinge region that, in principle, promotes the movement of the carboxybiotin.33) Furthermore, the highly conserved biotin-binding site, EVMKM, was located from residues 714 to 718 and represents the region of the BCCP domain. The binding sites for carboxybiotin and acetyl-CoA are also present in the CT domain at residues 1578–1628 and 1873–1892, respectively.

Comparative analysis of the primary structure of ACCs has demonstrated a highly conserved organization of the multidomains underpinning their relevant catalytic functions. With regard to sequence homology, the cloned gene from L. starkeyi resembles the fungal ACC involved in malonyl-CoA production. In addition, the absence of an N-terminal extension required for a canonical mitochondrial targeting signal indicated that the LsACC1 protein is categorized as a multi-domain ACC belonging to the small family of biotin-dependent carboxylase.

Accumulation of G3P by overexpression of GPD1 in S. cerevisiae did not improve lipid accumulation

As we know, G3P is a key intermediate in biosynthesis of TAG, and YA103 strain where GPP activity is completely abolished (deletion of GPP1 and GPP2) has been already shown to accumulate G3P.23) So we used the YA103 strain bearing the empty vector pX2-GAL (YA103g strain) as a control instead of the untransformed strains in order to enable direct comparison with the same strains bearing the plasmid pX2-GPD1 for GPD1 overexpression (YA103p strain). QPCR analysis of the transgenic yeasts confirmed fivefold expression levels of GPD1 to control levels (Fig. 3(A)). An obvious increase (1.5-fold) of G3P was obtained in strain YA103p compared with YA103g (Fig. 4). Our results were similar to Nguyen et al.34) To determine the influence of accumulation of G3P on cell growth and lipid accumulation in YA103, the DCW and lipid productivity of YA103g and YA103p were compared in SD-L medium. However, no differences concerning the lipid content could be detected between both strains (Table 2), most probably due to the lack of a concomitant excess of fatty acids, the second component needed for TAG synthesis.

Fig. 3. Relative expression levels of GPD1 and ScACC1 in transgenic yeasts.

Notes: (A) The estimation of relative expression levels was based on 2−ΔΔCT, where ΔΔCT = (CT gene of interest—CT internal control) overexpressing GPD1 gene of YA103p, YA103pl and YA103ps—(CT gene of interest—CT internal control) control YA103 g. (B) The estimation of relative expression levels was based on 2−ΔΔCT, where ΔΔCT = (CT gene of interest—CT internal control) overexpressing ScACC1 gene of YA103ps—(CT gene of interest—CT internal control) control YA103g. CT represents the cycle number at which a sample reaches a predetermined threshold signal value for the specific target gene. All the experiments were performed in triplicate.
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Fig. 4. Intracellular accumulation of G3P in transgenic yeasts.

Notes: Cells were cultivated in SD-L in shake flasks and harvested for extraction of G3P in the mid-log phase of growth. Data shown are the average values from three independent experiments including the standard deviations.
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Table 2. Lipid content, lipid productivity, and DCW of transgenic S. cerevisiae.

This hypothesis was experimentally proven when YA103g and YA103p were cultured on SD-L medium by feeding external oleic acid (Table 2). The YA103p overexpressing GPD1 gene led to a lipid content of 13.0%, a 37% increase from a lipid content of 9.5% in the background strain YA103g. Notably, lipid productivity also increased from 292.5 to 340 mg/L. So, in the S. cerevisiae, the fatty acids were the main element for TAG synthesis rather than G3P. We can overexpress the ACC gene which was the rate-limiting enzyme in fatty acid biosynthesis and increase total lipid in YA103.

LsACC1 expression analysis and overexpression of the gene in S. cerevisiae

The yeast L. starkeyi AS 2.1560 was cultured in a nitrogen-deficient medium with a C/N molar ratio of 160. The time courses of DCW and the lipid content of the bath culture are shown in Fig. 5(A). DCW and lipid contents for the 12-h, 48-h, and 96-h samples were 0.55 g/L and 17.3%, 7.21 g/L and 33.8%, and 12.97 g/L and 45.3%, respectively. Our findings indicate that the 12-h sample was suitable to represent the early stage of lipid production, and the 48-h sample was selected to represent the middle stage of lipid production. Finally, the 96-h sample was used for the late stage. The expression profiles of LsACC1 during the lipid fermentation process were analyzed by the qPCR method. The expression level of 48-h sample was fivefold higher than the 12-h sample. However, the mRNA level dropped in the 96-h sample (Fig. 5(B)). QPCR analysis of LsACC1 showed that the expression levels were upregulated during 48-h sample, which was consistent with the rapid accumulation of lipid. Liu et al. 35) reported that the expression of LsACC1 was upregulated during the lipid accumulation progress according to proteome analysis of L. starkeyi. For the experiment results, we supposed that the expression levels of the LsACC1 increased along with the lipid accumulation, because the gene is first committed step in fatty acid biosynthesis. However, in the last stage of lipid accumulation, the abundance of LsACC1 was enough for fatty acid biosynthesis, so the expression levels of mRNA decreased quickly.

Fig. 5. The lipid content and DCW during cell growth of L. starkeyi AS 2.1560 in the nitrogen-deficient medium and qPCR analysis of LsACC1 expression in 12-h, 48-h, and 96-h samples.

Notes: (A) Symbols: ■ denotes the lipid content (%); ● denotes the accumulation of DCW. (B) The estimation of relative expression levels was based on 2−ΔΔCT, where ΔΔCT = (CT gene of interest—CT internal control) overexpressing LsACC1 gene of 48-h and 96-h—(CT gene of interest—CT internal control) control 12-h. CT represents the cycle number at which a sample reaches a predetermined threshold signal value for the specific target gene. All the experiments were performed in triplicate.
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The conversion of acetyl-CoA to malonyl-CoA by the ACC was the rate-limiting step of fatty acid biosynthesis in many organisms including yeast.36) To test the function of LsACC1, we overexpressed LsACC1 in S. cerevisiae to increase the pool of malonyl-CoA for biosynthesis fatty acyl-CoA. Plasmid-based overexpression of LsACC1 led to a lipid content of 7.3%, a 24% increase from a lipid content of 5.9% in the YA103 g strain (Table 2). Notably, total lipid productivity also increased from 235 to 287.5 mg/L. This outcome proved the enzyme activity of the gene.

Heterologous expression of ACC with GPD1 gene in the S. cerevisiae YA103

To compare the ability of LsACC1 and ScACC1 gene to regulate fatty acids and lipid biosynthesis in S. cerevisiae, the two mutants YA103pl (transformed with pX2-GPD1 and pLSA1.0) and YA103ps (transformed with pX2-GPD1 and pSCA1.0) were transformed, respectively. The expression level of ScACC1 in YA103ps was fourfold higher than the control strain YA103g (Fig. 3(B)). Meanwhile, the expression level of GPD1 in both transformants is both more than fivefold than YA103g (Fig. 3(A)), and the lipid contents were significantly higher than that of YA103p, which were a result of the overexpression of the LsACC1 and ScACC1 under the control of the GAL1 promoter (Table 2). However, the lipid content of two ACC-overexpressing YA103pl and YA103ps showed no significant differences (Table 2), most likely due to differences in codon usage between L. starkeyi and S. cerevisiae. Meanwhile, the feed of external oleic acid can accumulate more lipid than overexpression of LsACC1 in the yeast YA103p.

In general, overexpression of LsACC1 in YA103p showed an overall enhancement in lipid production, where the highest accumulation resulted in a 63% increase in total lipid content (from 5.9 to 9.6%), so the gene can yield malonyl-CoA for acyl-CoA synthesis and subsequently increase the TAG synthesis. This result is similar to previous reports found in fungi, plants, and E. coli. In fungi, the heterologous expression of an oleaginous fungus, Mucor rouxii ACC in a non-oleaginous yeast, Hansenula polymorpha showed a 40% increase in total fatty acid content.18) In addition, targeting of the homomeric Arabidopsis ACC to plastids of Brassica napus resulted in an increase in the enzyme activity by approximately 20-fold with only a 5% increase in the total oil content of the seeds.17) It has been also demonstrated that the overexpression of the ACC in E. coli led to a threefold increase, and co-expression of ACC and malic enzyme resulted a 5.6-fold increase in intracellular lipids.37)

Taken together, we overexpressed the LsACC1 gene in YA103p led to a lipid content of 9.6%, but it was still low in contrast to the oleaginous yeast. Therefore, the low levels of lipid content found in the S. cerevisiae transformant may be explained by constraints of other metabolic processes involved in lipid accumulation in addition to acetyl-CoA carboxylation.

Funding

This work was financially supported by the Chinese National Key Technology R&D Program [2015BAD15B02] and the NSFC [31300569].

Disclosure statement

No potential conflict of interest was reported by the authors.

Acknowledgment

We would like to thank Prof. Anna-Karin Påhlman (Göteborg University, Göteborg) for kindly providing the YA103 mutant.

Notes

Abbreviations: ACC, acetyl-CoA carboxylase; TAG, triacylglycerol; G3P, glycerol 3-phosphate; GPD, glycerol 3-phosphate dehydrogenase; DCW, dry cell weight; DHAP, dihydroxyacetone phosphate; GPP, glycerol 3-phosphatase.

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