Reengineering lipid biosynthetic pathways of Aspergillus oryzae for enhanced production of γ-linolenic acid and dihomo-γ-linolenic acid

Accepted Manuscript Reengineering lipid biosynthetic pathways of Aspergillus oryzae for enhanced production of γ-linolenic acid and dihomo-γlinolenic acid

Sukanya Jeennor, Jutamas Anantayanon, Sarocha Panchanawaporn, Sakda Khoomrung, Chanikul Chutrakul, Kobkul Laoteng PII: DOI: Reference:

S0378-1119(19)30432-9 https://doi.org/10.1016/j.gene.2019.04.074 GENE 43833

To appear in:

Gene

Received date: Revised date: Accepted date:

28 February 2019 18 April 2019 26 April 2019

Please cite this article as: S. Jeennor, J. Anantayanon, S. Panchanawaporn, et al., Reengineering lipid biosynthetic pathways of Aspergillus oryzae for enhanced production of γ-linolenic acid and dihomo-γ-linolenic acid, Gene, https://doi.org/10.1016/ j.gene.2019.04.074

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ACCEPTED MANUSCRIPT Reengineering lipid biosynthetic pathways of Aspergillus oryzae for enhanced production of -linolenic acid and dihomo--linolenic acid

Sukanya Jeennor1  Jutamas Anantayanon1  Sarocha Panchanawaporn1  Sakda

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Khoomrung2,3  Chanikul Chutrakul1  Kobkul Laoteng1

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 Kobkul Laoteng

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Tel: +66-2-5646700, Fax: +66-2-5646704

Functional Ingredients and Food Innovation Research Group, National Center for Genetic

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E-mail: [email protected]

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Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong

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Luang, Pathum Thani, 12120, Thailand

Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University,

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Bangkok, 10700, Thailand

Siriraj Metabolomics and Phenomics Center, Faculty of Medicine Siriraj Hospital, Mahidol

University, Bangkok, 10700, Thailand

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ACCEPTED MANUSCRIPT ABSTRACT Biological significance of 18-carbon polyunsaturated fatty acids, -linolenic acid (GLA; C18:3 n-6) and dihomo--linolenic acid (DGLA; C20:3 n-6) has gained much attention in the systematic development of optimized strains for industrial applications. In this work, a n-6

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PUFAs-producing strain of Aspergillus oryzae was generated by manipulating metabolic reactions in fatty acid modification and triacylglycerol biosynthesis. The codon-optimized

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genes coding for 6-desaturase and 6-elongase of Pythium sp., and diacylglycerol

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acyltransferase 2 (mMaDGAT2) of Mortierella alpina were co-transformed in a single vector

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into A. oryzae BCC14614, yielding strain TD6E6-DGAT2. Comparative phenotypic analysis showed that a 70% increase of lipid titer was found in the engineered strain, which was a result

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of a significant increase in triacylglycerol (TAG) content (52.0 ± 1.8% of total lipids), and corresponded to the increased size of lipid particles observed in the fungal cells. Interestingly,

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the proportions of GLA and DGLA in neutral lipids of the engineered strain were similar, with

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the highest titers obtained in the high C:N culture (29:0; 6% glucose) during the lipidaccumulating stage of growth. Time-course expression analysis of the engineered strain

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revealed transcriptional control of TAG biosynthesis through a co-operation between the native

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DGAT2 of A. oryzae and the transformed mMaDGAT2.

Keywords: Aspergillus oryzae  Diacylglycerol acyltransferase  Dihomo-gamma-linolenic acid  Lipid accumulation  Triacylglycerol  Oleaginous fungi

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ACCEPTED MANUSCRIPT 1. Introduction Gamma-linolenic acid (GLA, C18:3 6,9,12) and dihomo--linolenic acid (DGLA, C20:3 8,11,14) are long-chain polyunsaturated fatty acids (LC-PUFAs) of the n-6 series, which are precursors for the biosynthesis of a biologically active compound, called prostaglandin E1

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(PGE1) (Fan and Chapkin, 1998; Wang et al., 2012). It has been reported that the n-6 PUFAs play crucial roles in human and animal health, such as anti-inflammation. Moreover, DGLA

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also has anti-proliferative properties (Nakamura et al., 1993; Nasrollahzadeh et al., 2008). In

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the past decades, many efforts have been made to study oleaginous microorganisms as alternatives for the production of functional lipids. Several oleaginous species such as Mucor

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circinelloides are able to synthesize GLA (Al-Hawash et al., 2018; Qiao et al., 2018). However,

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a limited number of DGLA-producing strains has been found, including Mortierella alpina (Jareonkitmongkol et al., 1992) and Lobosphaera incisa (Abu-Ghosh et al., 2015), although

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the DGLA contents in such oleaginous strains are very low due to the fact that this 20C-PUFA

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is an intermediate for the biosynthesis of other longer fatty acyl chains. Hence, strain improvement of oleaginous microbes has been postulated as a potential strategy for enhancing the production of n-6 PUFAs (Abu-Ghosh et al., 2015; Chutrakul et al., 2016;

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Jareonkitmongkol et al., 1993; Yazawa et al., 2007). The ‘cell chassis’ concept is an important

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method for performing metabolic engineering based on differences in genotypic and phenotypic characteristics among oleaginous microbes (Thevenieau and Nicaud, 2013). Even in the same oleaginous species, a discrimination in fatty acid and lipid profiles was found depending on cultivation conditions, and growth and developmental stages (Cheawchanlertfa et al., 2011; Jeennor et al., 2006; Laoteng et al., 2008). Based on fatty acid profiles, the oleaginous fungus, Aspergillus oryzae, has been exploited as a host system, aiming to generate a DGLA-producing strain by overexpressing 6-desaturase and 6-elongase genes of Pythium for the bioconversion of endogenous linoleic acid (LA; C18:2 9,12) to GLA and DGLA, 3

ACCEPTED MANUSCRIPT respectively (Chutrakul et al., 2016). However, in that study the DGLA proportion in total lipid extracted from the recombinant strain was very low (1.8-2.0% of total lipid), and it was preferentially distributed into phospholipid (PL) rather than neutral lipid (NL) fractions. Typically, oleaginous strains store lipids in their cells in a special organelle, termed the

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lipid particle (LP) or lipid body (LB). Triacylglycerol (TAG) is predominant in the storage lipids and not only serves as an energy source but also functions to sequester harmful lipids in

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the cells, such as free fatty acids and some unusual fatty acids (Athenstaedt and Daum, 2006).

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In eukaryotic organisms, TAG is synthesized via the acyl-CoA-dependent Kennedy pathway, in which diacylglycerol acyltransferase enzyme (DGAT) plays a key role in the rate-limiting

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step of transferring an acyl group to diacylglycerol (DAG) (Czabany et al., 2007). On the

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contrary, phospholipid:diacylglycerol acyltransferase (PDAT) catalyzes the transfer of a fatty acyl chain from phospholipids to DAG at the sn-3 position through an acyl-CoA-independent route (Beopoulos et al., 2008; Czabany et al., 2007; Daum et al., 2007). Three families of

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DGAT enzymes have been identified. DGAT1 and DGAT2, responsible for TAG biosynthesis, are ER-localized enzymes (Turchetto-Zolet et al., 2011), whereas DGAT3 is localized in the cytosol. It has been reported that the affinity of DGAT2 enzymes for their substrates was

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stronger compared with that for DGAT1 enzymes (Yen et al., 2008).

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For the production of particular fatty acids with industrial importance, the type of fatty acid constituents in TAG should be considered in addition to the production yields and titers. Thus, a DGAT2 enzyme specific for a targeted fatty acid substrate is of particular interest in this respect. The OtDGAT2 enzyme of microalga Ostreococcus tauri was characterized, showing its broad substrate specificity for saturated fatty acids, and mono- and polyunsaturated fatty acyl-CoA (Wagner et al., 2010). Recently, the substrate specificity of the MaDGAT2 enzyme from the 20C-PUFA producing species, M. alpina, was studied by heterologous expression of the encoding gene in Saccharomyces cerevisiae (Jeennor et al., 4

ACCEPTED MANUSCRIPT 2017). It was found that n-6 PUFAs, particularly DGLA and GLA, were preferred fatty acyl substrates for the MaDGAT2 enzyme. In the present study, we aimed to enhance the production of GLA and DGLA in A. oryzae by engineering lipid biosynthetic pathways through genetic and physiological manipulations. The metabolic reactions in fatty acid modification (desaturation and elongation) and TAG biosynthesis were targets for engineering the fungal

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strain. Time-course analysis of cell growth, fatty acid composition and lipid content of the

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engineered strain was performed. In addition, transcriptional expression analysis of native and

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transformed DGAT genes at different growth stages was implemented to investigate how the

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fungal cell acclimatizes to the transgenes.

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2. Materials and methods 2.1 Strains and cultivations

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Aspergillus oryzae strain BCC14614 was used as a host system and routinely cultivated

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on Potato Dextrose Agar (PDA) medium (BD, USA) at 30 C for 5 days. For preparation of spore inoculum, the untransformed parent and engineered strains were individually grown on

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polished rice at 30 C for 5 days. Then, spores were harvested by using 0.01% (v/v) Tween 80 solution and filtered through Miracloth (Merck, Germany). The spore suspension was added

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into 50 mL culture medium to obtain the final concentration of 106 spores/mL. The semisynthetic medium (SM) (Laoteng et al., 2008) was used for fungal cultivation for primary lipid analysis. For further phenotypic characterization at different growth stages, fungal cells were grown in the production medium (PM), which consisted of 0.3% (w/v) yeast extract, 0.3% (w/v) NaNO3, 0.1% (w/v) K2HPO4, 0.05% (w/v) MgSO4, 0.05% (w/v) KCl, 0.001% (w/v) FeSO4 and variable glucose concentrations. PM media with two C:N ratios, 14.5 and 29.0,

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ACCEPTED MANUSCRIPT contained 3% and 6% glucose (w/v), respectively. All cultures were incubated at 30 C with agitation at 250 rpm. Saccharomyces cerevisiae strain INVSCI (MAT, his3-1, leu2, trp1-289, ura3-52, MAT, his3-1, leu2, trp1-289, ura3-52) purchased from Invitrogen (CA, USA) was used for DNA assembly. For DNA transformation, the yeast culture was incubated at 30 C with shaking of 200

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rpm to obtain a cell density of 107 cells/mL. The selection of yeast transformants was done by

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cultivation on a minimal medium (SD), which consisted of 0.67% (w/v) bacto-yeast nitrogen

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base without amino acids, 2% (w/v) glucose, and three amino acids, L-histidine-HCl, Ltryptophane and L-leucine at the concentrations of 20, 20 and 30 mg/L, respectively.

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Escherichia coli strain DH5αsupE44, lacU169, (80, lacZM15), hsdR17, recA1,

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endA1, gyrA96, thi1,relA1 was employed for propagation of recombinant plasmid. It was

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cultivated at 37 °C in Luria-Bertani medium (LB) containing 100 mg/L ampicillin.

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2.2 Plasmid construction for co-expression of the genes involved in PUFA and TAG biosynthesis in A. oryzae

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The recombinant plasmid containing three genes, encoding DGAT2, 6-desaturase and 6-elongase, was constructed using the DNA assembly technique in S. cerevisiae

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(Pahirulzaman et al., 2012) as shown in Fig. 1. The pAMDes6-MElo6 vector backbone containing the codon-optimized 6-desaturase (MPyDes6) and 6-elongase (MPyElo6) genes of Pythium sp. (Chutrakul et al., 2016) was restricted with EcoRI and then subjected to additional insertion of the codon-optimized gene of M. alpina (mMADGAT2) coding for the PUFA-specific DGAT2 enzyme (Jeennor et al., 2017). The enolase promoter (Penol) sequence (0.6-kb fragment) and mMaDGAT2 gene (1.0-kb fragment) were amplified using the plasmid BPO140 and PymMaDGAT2 (Jeennor et al., 2017) as templates, respectively. The primers

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ACCEPTED MANUSCRIPT (Penol-F and Penol-R) used for amplifying the Penol promoter were designed for sequence overlapping of the nopaline synthase terminator (Tnos3) and the 5-portion of mMaDGAT2 gene, respectively (Supplementary Table S1). For amplification of the mMaDGAT2 gene, the specific primers, mMaDGAT2-F1 and mMaDGAT2-R1, were designed for sequence overlapping between Penol promoter and the glyceraldehyde-3-phosphate dehydrogenase

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(PgpdA) promoter, respectively. Polymerase chain reaction (PCR) was performed using

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Platinum Taq Hi-fi DNA polymerase (Invitrogen, CA) following the manufacturer’s

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instruction. The DNA amplicons were purified using QIAEXII kit (QIAGEN, Germany). The two amplified fragments and the EcoRI-restricted backbone vector were co-transformed into

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the yeast cells by the lithium acetate/PEG method according to the Invitrogen™ user manual. Rapid investigation of the DNA assembly in yeast transformants was conducted by PCR using

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Taq DNA polymerase (Thermo Scientific, USA). Plasmid extraction was performed using Yeast Plasmid Miniprep (Zymoprep™, USA). The obtained plasmid was shuttled to E. coli for

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its propagation, and subsequently confirmed by enzyme restriction analysis and DNA

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sequencing.

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2.3 Fungal transformation

Protoplasts of A. oryzae were prepared following a modified method (Chutrakul et al.,

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2013). The protoplasts were suspended in ice-cold sorbitol/Tris-HCl/CaCl2 solution (STC) and then subjected to transformation with 5 µg of the recombinant plasmid by the PEG-mediated method. The transformed protoplasts were regenerated in Potato Dextrose Broth (PDB) containing 1.2 M sorbitol for 2 h, immediately plated on Czapek Dox medium (Difco, USA) with 175 g/mL phleomycin, and were incubated at 25 C for 7 days. Phleomycin-resistant colonies were selected for further analysis of the presence of the Ble gene in the cells by PCR using Taq DNA polymerase (ThermoScientific) and the specific primers (Ble-466-F and Ble-

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ACCEPTED MANUSCRIPT 466-R) (Supplementary Table S1). The selected transformants were subcultured five times alternately on selective and non-selective PDA media to derive genetically stable clones. The resulting stable transformant of A. oryzae was designated as strain TD6E6-DGAT2.

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2.4 Determination of biomass and glucose concentration Growth profiles of the engineered strain and untransformed parent were determined.

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Mycelial cells were harvested at different time points by filtration with gentle suction and

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then were dried in a hot-air oven at 60 C until the weight was constant. Cell biomass was

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thus measured as dry cell weight (DCW).

The culture broth was filtered through 0.2 µm sterile filter and then subjected to glucose

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analysis using high-performance liquid chromatography (HPLC; Ultimate 3000, Thermo, USA) equipped with refractive index detector and Aminex HPX-87H ion exclusion column

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(Bio-Rad Laboratories, Hercules, CA, USA). Chromatographic separation was performed

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using the isocratic mode. The mobile phase used was 5 mM H2SO4 solution at a flow rate of 0.6 mL/min at 60 C for 30 min. Quantitative analysis of residual glucose in the culture broths

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mg/mL.

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was performed using a calibration curve of sugar standard in a concentration range of 0.1-10.0

2.5 Lipid particle measurement LPs in the fungal cells were assessed as previously described (Kimura et al., 2004). 10 µL of dimethyl sulfoxide (DMSO) were added into the fungal culture samples harvested at different time points. Cell staining was performed by adding 5 µL Nile Red dye (0.01 mg/mL in acetone) into the samples. After incubation in the dark for 10 min, the LPs were visualized

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ACCEPTED MANUSCRIPT with fluorescence microscopy (Olympus system microscope BX53E-DP73) with excitation at 470-495 nm, and emitted light was collected at 510 nm. Approximately, 30 LPs from each sample were analyzed to measure average diameter.

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2.6 Intracellular lipid and fatty acid analyses

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Total lipid (TL) extraction was performed according to the standard method (Folch et al.,

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1957). Lipid contents were weighed and expressed as percentage of DCW (% lipid/DCW, w/w). Lipid classes were quantified by HPLC (Ultimate 3000, Thermo, USA) equipped with

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charged aerosol detector (CAD) (Corona; ESA, USA). The lipid sample (10 mg/mL) was injected into the Luna 5 µm HILIC 200A column (250  4.6 mm, Phenomenex, USA) at 35

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C. The HPLC chromatographic conditions for separation and the detection parameters by CAD were set according to the published protocol (Khoomrung et al., 2013). Individual lipids

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were identified by comparing their retention times with those of authentic lipid standards

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(Sigma, USA) under identical measurement conditions. High performance-thin layer chromatography (HP-TLC) was performed to separate

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individual lipid classes for further fatty acid analysis. In brief, TL was solubilized in

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chloroform:methanol solution (2:1, v/v) and then applied on a silica plate (HP-TLC silica gel plate 60F 254; CAMAG, Switzerland). The separation was performed in a solution containing hexane, diethyl ether and acetic acid (70:30:1, v/v) (Certik et al., 1996). The separated lipid classes were visualized by iodine vapor application (Sajbidor et al., 1994) and identified by comparison of their Rf values with those of authentic standards. Subsequently, the bands corresponding to TAG, steryl ester (SE), PLs and free fatty acid (FFA) were immediately scraped off for analyzing total fatty acids (TFA).

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ACCEPTED MANUSCRIPT Fatty acid methyl esters (FAMEs) were prepared using a modified method as previously reported (Chutrakul et al., 2016) and analyzed by gas chromatography equipped with flame ionization detection (Agilent 7890B, California, USA). The column was a HP-88 capillary column (100 m  0.25 mm I.D.  0.20 µm film thickness, Agilent, USA). Individual fatty acids were identified by comparing their retention times with those of FAME standards (Sigma,

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USA). Using pentadecanoic acid (C15:0) as an internal standard, fatty acid contents were

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quantified by calculating their chromatographic peak areas.

reverse transcription PCR (RT-qPCR)

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2.7 Expression analysis of DGAT genes in the engineered strain of A. oryzae by quantitative

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Based on the conserved characteristics of the DGAT2 family (Cao, 2011), the putative DGAT2 sequences of A. oryzae were retrieved from the GenBank database (NCBI,

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http://www.ncbi.nlm.nih.gov) using the MaDGAT2 sequence of M. alpina as a query. The

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identified gene sequences of A. oryzae (AoDGAT2I and AoDGAT2II) were used for primer design for transcriptional analysis.

The TD6E6-DGAT2 transformant and untransformed BCC14614 cultures were grown

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in PM medium containing 6% glucose (w/v) at 30 C and harvested at different time points.

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Total RNA extraction was performed using PureLinkTM RNA mini kit (Life Technologies, USA). Transcript levels of the two native DGAT2 genes of A. oryzae (AoDGAT2I and AoDGAT2II) and the heterologous gene of M. alpina DGAT2 (mMaDGAT2) were quantified by RT-qPCR using Sso AdvancedTM universal SYBR® Green super-mix (Bio-Rad, USA) with Bio-Rad CFX96TM Real-Time PCR Detection System. Specific primers for amplifying individual genes are shown in Supplementary Table S1. Approximately 1 g of total RNA was subjected to reverse transcription using an oligo-dT primer and the SuperScript II first-strand synthesis system (Invitrogen, CA, USA) according to the manufacturer’s instructions. 18S 10

ACCEPTED MANUSCRIPT rRNA was used as a normalization reference for measuring expression ratios of the targeted genes. The cycle threshold (CT) values were determined and the relative fold difference was calculated by using the 2− CT method (Livak and Schmittgen, 2001). Individual samples were

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analyzed in triplicate.

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2.8 Data analysis

All experiments were conducted in triplicate independently, and the resulting data

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were presented as means with standard deviation. Statistical analysis of data was done using

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the Statistical Package for Windows® and considered statistically significant at P <0.05.

2.9 Nucleotide sequence accession numbers

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The codon-optimized sequences of MPyDes6, MPyElo6 and mMaDGAT2 genes have

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been deposited in GenBank under the accession numbers KT438838, MK091394 and KY859195, respectively. The native DGAT2 genes of A. oryzae, AoDGAT2I and AoDGAT2II,

3. Results

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respectively.

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have been deposited in GenBank under the accession numbers EIT83121 and OOO08321,

3.1 GLA- and DGLA-producing strain of A. oryzae generated by co-expression of three genes involved in fatty acid modification and TAG biosynthesis Using the yeast assembly technique, the expression plasmid, pAMD6E6-DGAT2 (16.1 kb) was generated by such that the codon-optimized genes of mPyDes6, mMaDGAT2 and 11

ACCEPTED MANUSCRIPT mPyElo6 were located downstream of promoters PgpdA, Penol, and PtoxA, respectively (Fig. 1). After plasmid propagation in E. coli, the vector was extracted and analyzed by EcoRI-PacI restriction and DNA sequencing. It was shown that the mMaDGAT2 sequence was introduced in-frame into the pAMDes6-MElo6 vector. After introducing the pAMD6E6-DGAT2 plasmid into A. oryzae cells, positive transformants selected on the phleomycin-containing medium

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were analyzed by PCR analysis. The results showed that the 0.47-kb fragment of the Ble

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sequence was found in the selected stable clone (TD6E6-DGAT2) after alternate subculturing

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five times with and without phleomycin selection (Supplementary Fig. S2). In addition, DNA fragments corresponding to mMaDGAT2, MPyDes6 and MPyElo6 were also detected by PCR

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in these fungal cells, indicating stability of the transformed heterologous genes (Supplementary Fig. S3).

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Primary lipid analysis of the engineered strain was done by growing it in SM medium at 30 C for 72 h. The results showed that GLA and DGLA (10.2±0.1 and 4.1±0.1 % of total

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fatty acid, respectively) were detected in the TD6E6-DGAT2 culture as a result of the

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expressed 6-desaturase and 6-elongase enzymes. Under the same culture conditions, the GLA and DGLA proportions of strain TD6E6-DGAT2 were comparable to those of the

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previously constructed strain TD6E6 without the mMaDGAT2 gene (Chutrakul et al., 2016), in which 10.4 ±0.4 % GLA and 5.0±0.2 % DGLA of total fatty acid were obtained. As expected,

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the total lipid content of strain TD6E6-DGAT2 (16.7±0.2% total lipid in DCW) was higher than that of the untransformed and TD6E6 strains (12.8±0.7% and 13.6±0.5% total lipid in DCW, respectively).

3.2 Characterization of biomass and lipid production in strain TD6E6-DGAT2 3.2.1 Comparative growth profiles of the TD6E6-DGAT2 and untransformed strains

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ACCEPTED MANUSCRIPT To further characterize cell growth and biomass production of the TD6E6-DGAT2 strain, the fungal cells were cultivated in the production media with low glucose (3%, w/v) and high glucose (6%, w/v) concentrations. Similar growth profiles were observed for the TD6E6DGAT2 and untransformed strains (Fig. 2). In addition, there was no significant difference (P>0.05) in biomass production between the two strains, in which biomass contents rapidly

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increased for the first 48 h (Fig. 2a and 2b), and coincided with the sharp reduction of residual

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glucose concentrations in the fermented broths. After glucose exhaustion, the biomass

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gradually decreased, particularly in the 3% glucose culture. The maximal biomass titers were obtained in the 48-h cultures containing 3% glucose (Fig. 2a), whereas prolonged cultivation

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time was required to reach the maximum biomass levels of the both strains when using 6% glucose (Fig. 2b). As such, the biomass productivities (0.25-0.26 g/L/h) of the individual strains

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using different glucose concentrations were not different.

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3.2.2 Lipid phenotypes of the TD6E6-DGAT2 strain with different growth conditions Using Nile red staining, LPs were visualized during cell growth of the engineered strain

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TD6E6-DGAT2 and the untransformed parent strain of A. oryzae and clearly observed at late logarithmic phase (Fig. 3). However, LPs from the TD6E6-DGAT2 cultures were larger than

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those from the untransformed strain (P<0.05) when grown in PM medium containing 6% (w/v) glucose. The maximal LP diameter was found in the engineered culture grown for 72 h. Using 3% (w/v) glucose, a significant difference in lipid particle sizes between the TD6E6-DGAT2 and untransformed cultures was also detected at late logarithmic phase (48 h), however prolonged cultivation (72 h) without glucose in the culture system did not enhance their LP sizes. Further analysis of intracellular lipids showed that the lipid contents of the untransformed and TD6E6-DGAT2 cultures increased continuously with cell growth. The 13

ACCEPTED MANUSCRIPT maximal lipid contents were observed in 3% and 6% glucose cultures at the late growth stage, called the lipid-accumulating phase, which were grown for 48 and 72 h, respectively (Fig. 2c and 2d). Although the characteristics of growth and lipid accumulation of the TD6E6-DGAT2 culture were similar to the untransformed parent, the lipid content and titer of the engineered culture were markedly enhanced by using 6% (w/v) glucose, accounting for 53% and 70%

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increases, respectively, as compared with those of the untransformed culture (Fig. 2 and Table

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1).

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HPLC-CAD analysis of the 6% (w/v) glucose cultures revealed that NLs (TAG and SE) were highly accumulated in the fungal cells harboring mMDGAT2 during the lipid-

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accumulating phase (72 h) as shown in Fig. 4, and were positively correlated with the size of cellular LPs. When compared with the untransformed parent, about 2.6-3.0 fold increase of

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TAG contents was observed in the TD6E6-DGAT2 cultures corresponding to the enhanced lipid contents (Table 1). In contrast, PL and FFA proportions in total lipid of the engineered

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strain were lower than those of the untransformed parent (Fig. 4). Considering fatty acid

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composition in individual lipid classes of the cultures grown in the high C:N medium, the proportion of LA in all lipid classes of the TD6E6-DGAT2 culture markedly decreased as a

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result of the accumulated 6-desaturated and 6-elongated products (GLA and DGLA) when compared with the untransformed parent. The triene fatty acids, GLA and DGLA, of the

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engineered strain were found in similar proportions in almost all lipid classes with the exception of PL. The distribution of GLA in PL was higher than that of the 20C-PUFA. Obviously, the maximal titers of GLA and DGLA were obtained in the TD6E6-DGAT2 culture grown in the 6% glucose medium for 72 h (Table 1).

3.3 Transcriptional expression of native and heterologous DGAT2 genes in the engineered strain 14

ACCEPTED MANUSCRIPT To investigate the transcriptional control of TAG biosynthesis in the TD6E6-DGAT2 strain, the identification of native DGAT2 genes of A. oryzae was undertaken. BLAST analysis using the DGAT2 sequence of M. alpina as a query showed that there were two putative DGAT2 sequences in A. oryzae (AoDGAT2I and AoDGAT2II), which contained conserved motifs shared among DGAT2 sequences of several fungal species (Supplementary Fig. S1).

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Thus, the expression of the native DGAT2 genes (AoDGAT2I and AoDGAT2II) in the 6%

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glucose cultures was investigated using RT-qPCR. In the untransformed parent, the highest

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transcript level of AoDGAT2I was found during the active growth phase (24-h culture) and then sharply reduced in contrast to the AoDGAT2II transcript, which slightly increased during the

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entire cell growth (Fig. 5). Interestingly, the transcript profiles of the two native DGAT2 genes in the engineered A. oryzae strain were altered when compared to the untransformed parent

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profiles. In the engineered strain (TD6E6-DGAT2), there was a 20-fold increase in the AoDGAT2I transcript level during the lipid-accumulating phase (72-h culture). A similar trend

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was also observed in the transcript profile of the heterologous gene (mMaDGAT2), where the

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expression level was up-regulated by a 4.0-fold increase. Notably, a slight decrease of AoDGAT2II transcript levels was detected during the entire cell growth of the engineered

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strain.

4. Discussion

The filamentous fungus, A. oryzae, exhibits superior performance for single cell oil production based on its oleaginous phenotype and nutrient assimilation (Ahmad et al., 2015; Muniraj et al., 2013). These phenotypic characteristics result from the efficient machinery elements governing lipid over-production, such as precursor generation, fatty acid synthesis and the lipid accumulation process (Thammarongtham et al., 2018; Vorapreeda et al., 2012,

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ACCEPTED MANUSCRIPT 2013). Thus, the improvement of fatty acid constituents in the fungal cell through biotransformation processes is of particular interest in value creation for production of specialty lipids. The combinatorial manipulation targeting of fatty acid and lipid biosynthetic pathways of this oleaginous fungus was implemented in this work, demonstrating the enhancement of GLA and DGLA production yields in A. oryzae. The problems associated with low DGLA

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content observed in the previous study (Chutrakul et al., 2016) were overcome by co-

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expressing mMaDGAT2 in conjunction with the mPyDes6 and mPyElo6 genes. An increased

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DGLA content might be a result of the transformed mMaDGAT2 with a preference for DGLA as substrate in addition to its main function in TAG formation (Jeennor et al., 2017). Apart

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from the genetic approach, we also showed that the growth phase and glucose concentration also affected the lipid production yield and the DGLA proportion of TL in strain TD6E6-

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DGAT2. It has been documented that the lipid over-production process in oleaginous yeasts and fungi is usually triggered by nitrogen deprivation and carbon excess or in turn an optimal

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C:N ratio (Immelman et al., 1997; Koike et al., 2001; Ratledge, 2002). Noteworthy, the high

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concentration of glucose (6%, w/v) in this study enhanced both biomass and lipid production of the A. oryzae recombinant strain, eventually the glucose being completely consumed at the

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end of cell growth (Fig. 2). Interestingly, it seems that there was no lag phase of cell growth observed in the 6% glucose cultures. As such, how the cells tolerate high osmotic pressure

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should be further assessed to achieve the optimal nutrient concentrations for maximizing lipid productivity without the perturbation of cell growth and biomass production. Actually, acyltransferase reactions responsible for facilitating incorporation of the newly synthesized fatty acids into certain lipids are controlled by a set of enzymes, such as glycerol 3-phosphate acyltransferase, lysophosphatidate acyltransferase, phosphatidate phosphatase, PDAT and DGAT (Pillai et al., 1998). It has been well known that unsaturated fatty acids esterified in membrane phospholipids were the major substrates for desaturase and

16

ACCEPTED MANUSCRIPT elongase activities (Griffiths et al., 1988; Jackson et al., 1998). The normal growth found in the TD6E6-DGAT2 strain (Fig. 2) suggests that the fungal cells might have an acclimatization mechanism to cope with the highly unsaturated fatty acid products accounting for 16.3% of total lipid (Table 2). Possibly, the fatty acyl exchange between acyl-CoA and acyl-phospholipid pools might participate in lipid homeostasis of the engineered strain for maintaining membrane

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fluidity and cell function. The transcriptional responses of the native and heterologous DGAT2

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genes in the TD6E6-DGAT2 strain (Fig. 5) indicated that they acted in concert to control TAG

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biosynthesis. The substantial change in AoDGAT2I expression of the engineered strain during the lipid-accumulating phase, indicates that it might be a key regulatory gene involved in TAG

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formation of A. oryzae. Notably, low expression levels of AoDGAT2II were observed during the entire growth of the A. oryzae untransformed parent in contrast to the AoDGAT2I

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expression profile (data not shown). In addition, the enhanced LP size found in the engineered strain might be another responsive mechanism to prevent the toxicity of foreign triene fatty

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acids in the cells (Thevenieau and Nicaud, 2013; Yang et al., 2012). Taken together, this fungal

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platform system exhibited cell robustness to the altered genetic or in turn metabolic perturbation that elaborates the prospect for further multi-gene editing via emerging

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technologies for the production of specialty lipids with industrial interest. With an emphasis towards the channeling of GLA and DGLA to TAG for production

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improvement, the rate-controlling step of TAG biosynthesis was the target for pathway engineering by overexpression of mMaDGAT2 in A. oryzae. In the TD6E6-DGAT2 strain, the 6-desaturated 18C-fatty acid product (GLA) accumulated in NLs (TAG and SE) at a similar proportion to the 20-C PUFA product (DGLA). This might be explained by the substrate specificities of either the mMaDGAT2 or native DGAT2 enzymes of the host system. Although the mMaDGAT2 enzyme preferred DGLA, it was able to catalyze the transfer of other n-6 acyl substrates to TAG (Jeennor et al., 2017). Actually, this fungal host cell highly accumulates LA

17

ACCEPTED MANUSCRIPT as a major proportion of lipids, particularly in NLs (Table 2). These findings suggest that the substrate specificities of the native enzymes (AoDGAT2I and AoDGAT2II) and their regulatory mode should be further addressed. The microbial oils containing DGLA and GLA would provide health benefits according to their biological functions (Fan and Chapkin, 1998; Wang

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et al., 2012; Yi and Steven, 2014).

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5. Conclusion

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This work demonstrated how to improve the yield of DGLA accumulation in A. oryzae cells without the defects in growth and development. Through genetic and physiological

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manipulations of the relevant metabolic reactions, GLA and DGLA production yields were significantly enhanced by cultivating the engineered strain at optimal conditions, which would

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permit further process development for maximizing the productivity of high-value oils. Not only the nutrient-dependent regulation of the lipid accumulation process, but also controlling

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parameters of the fermentation process and the production costs should be taken into account

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to increase the feasibility and practicality of the production process for industrially viable

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applications.

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Compliance with ethical standards

Conflicts of interest

The authors declare there are no competing interests.

Author Contributions Sukanya Jeennor performed the experimental design, lipid classification, manuscript writing, figures and tables arrangement. Jutamas Anantayanon carried out fungal cultivation, 18

ACCEPTED MANUSCRIPT fatty acid analysis and gene expression analysis. Sarocha Panchanawaporn performed fungal transformation and transformant analysis. Sakda Khoomrung involved in lipid analysis and reviewed the manuscript. Chanikul Chutrakul carried out plasmid construction. Kobkul Laoteng contributed to the conception, execution, result interpretation, manuscript revision and

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completion of the final manuscript.

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Acknowledgements We are grateful to Mr. Witoon Youngsaad, Herb and Thai Traditional

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Medicine Division for technical assistance on HP-TLC technique and Ms. Mayura Veerana for transformant selection. We would like to thank Dr. Donald A. MacKenzie, Quadram Institute

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Bioscience, Norwich Research Park, Norwich, NR4 7UQ, UK and Dr. Eugene Fletcher for proof-reading. S.K is supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj

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Hospital, Mahidol University and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), the Office of the Higher Education Commission. This work was financially

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supported by Platform Research Grant (Grant No. P15-51082), National Center for Genetic

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glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. J. Lipid Res. 49, 22832301. https://doi.org/10.1194/jlr.R800018-JLR200

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Figure captions

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J. 37, 112–119. https://doi.org/10.4103/2319-4170.131378

cerevisiae.

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Fig. 1 Schematic construction of plasmid pAMD6E6-DGAT2 via DNA assembly in S.

Fig. 2 Cell growth (a, b) and lipid production (c, d) of the untransformed A. oryzae parent strain BCC14614 (gray-dotted line) and TD6E6-DGAT2 (black line) transformant. Cells were grown in PM media containing 3% (w/v) glucose (a, c) and 6% (w/v) glucose (b, d) at 30 C.

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ACCEPTED MANUSCRIPT Circle, asterisk, triangle and square symbols indicate cell biomass, residual glucose concentration, and lipid content and concentration.

Fig. 3 Lipid particles from the untransformed A. oryzae parent strain BCC14614and TD6E6-

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DGAT2 transformant grown in PM media containing 3% and 6% (w/v) glucose at 30 C for

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48 and 72 h. Nile Red-stained lipid particles (a) and the lipid particle diameter (b and c) of

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the values (P<0.01) are indicated by asterisks (**).

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the 48 h culture (black bars) and 72 h culture (grey bars) are shown. Significant differences in

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Fig. 4 Proportion of individual lipid classes in total lipid extracted from the untransformed parent (grey bars) and TD6E6-DGAT2 transformant (black bars) strains of A. oryzae. Cells

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were grown in PM medium containing 6% (w/v) glucose at 30 C for 48 (a) and 72 h (b).

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SE, sterol ester; TAG, triacylglycerol; FFA, free fatty acid; PL, phospholipid. Others indicate

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monoacylglycerol (MAG), diacylglycerol (DAG) and ergosterol (EG).

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Fig. 5 Relative expression of DGAT2 genes in A. oryzae strains quantified by RT-qPCR. Cells were grown in PM medium containing 6% (w/v) glucose. Time-course expression levels of native AoDGAT2I (a) and AoDGAT2II (b) genes in the untransformed parent strain BCC14614 are shown. Transcript levels of the native AoDGAT2I (c) and AoDGAT2II (d) genes, and the heterologous mMaDGAT2 gene (e) in the TD6E6-DGAT2 transformant strain are also illustrated. 18S rRNA was used as an internal control.

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ACCEPTED MANUSCRIPT Abbreviation list diacylglycerol acyltransferase, DGAT; dihomo-gamma-linolenic acid, DGLA; diacylglycerol, DAG; dry cell weight, DCW; ergosterol, EG; fatty acid methyl esters, FAMEs; free fatty acid, FFA; gamma-linolenic acid, GLA; lipid body, LB; long-chain polyunsaturated fatty acids, LClipid

particle,

LP;

monoacylglycerol,

MAG;

neutral

lipid,

NL;

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PUFAs;

phospholipid:diacylglycerol acyltransferase, PDAT; phospholipid, PL; polyunsaturated fatty

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acids, PUFAs; prostaglandin E1, PGE1; quantitative reverse transcription PCR, RT-qPCR;

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steryl ester, SE; triacylglycerol, TAG; total fatty acid, TFA; total lipid, TL

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ACCEPTED MANUSCRIPT Table 1 Lipid content, TAG content of dry cell weight, GLA and DGLA titers of the wild-type and TD6E6DGAT2 strains of A. oryzae. Cells were grown in CPM medium containing 6% (w/v) glucose at different time

GLA titer (mg/L)

DGLA titer (mg/L)

24

9.0 ± 0.5

2.3 ± 0.2

-

-

48

10.2 ± 0.1

2.5 ± 0.1

-

72

12.4 ± 1.8

4.0 ± 0.6

-

96

12.2 ± 0.8

3.4 ± 0.2

120

9.4 ± 0.8

2.6 ± 0.2

-

-

24

12.0 ± 0.4

5.3 ± 0.5

122.6 ± 7.5

45.5 ± 1.7

48

16.1 ± 1.6

7.5 ± 0.5

144.3 ± 14.8

86.0 ± 8.9

72

19.0 ± 1.4

10.2 ± 0.8

198.0 ± 8.7

166.5 ± 9.0

96

15.4 ± 0.8

7.2 ± 0.4

145.6 ± 5.1

154.5 ± 14.6

120

12.1 ± 1.1

6.8 ± 0.8

80.4 ± 13.4

69.1 ± 14.7

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TAG content (%, w/w)

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TD6E6DGAT2

Lipid content (%, w/w)

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Wild type

Cultivation time (h)

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Culture

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courses.

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Table 2 Fatty acid composition in individual lipid classes of the wild-type and TD6E6-DGAT2 strains. Cells were grown in CPM medium containing 6% (w/v) glucose at 30 C for 72 h.

Culture Wild type

TD6E6-DGAT2

a

T P

Fatty acid composition in total fatty acid (%, w/w)

Lipid class a

I R

C16:0

C16:1∆9

C18:0

C18:1∆9

C18:2∆9,12

C18:3∆6,9,12

C18:3∆9,12,15

C20:3∆8,11,14

TL

19.2 ± 0.4

1.1 ± 0.1

3.9 ± 0.0

12.6 ± 0.6

62.1 ± 0.8

0.0 ± 0.0

1.1 ± 0.2

0.0 ± 0.0

TAG

27.1 ± 2.0

3.6 ± 1.4

5.4 ± 0.0

9.0 ± 0.5

54.7 ± 0.1

0.0 ± 0.0

0.3 ± 0.0

0.0 ± 0.0

FFA

24.0 ± 4.6

6.9 ± 0.6

16.4 ± 1.0

19.9 ± 2.5

32.3 ± 0.5

0.0 ± 0.0

0.5 ± 0.0

0.0 ± 0.0

SE

22.4 ± 3.2

3.5 ± 0.5

5.8 ± 2.0

7.6 ± 0.0

60.4 ± 4.7

0.0 ± 0.0

0.3 ± 0.0

0.0 ± 0.0

PL

27.1 ± 2.0

3.6 ± 1.4

5.7 ± 0.0

9.0 ± 0.5

54.3 ± 0.1

0.0 ± 0.0

0.4 ± 0.0

0.0 ± 0.0

TL

21.3 ± 0.0

0.5 ± 0.0

8.9 ± 0.2

24.4 ± 1.3

28.1 ± 0.6

8.8 ± 0.5

0.5 ± 0.0

7.5 ± 0.3

TAG

24.9 ± 0.4

1.6 ± 0.1

10.4 ± 0.6

21.8 ± 0.2

26.0 ± 0.5

6.8 ± 0.4

0.8 ± 0.1

7.7 ± 0.2

FFA

31.7 ± 0.6

2.4 ± 0.3

11.2 ± 1.0

10.8 ± 1.1

26.6 ± 0.3

8.9 ± 0.2

2.2 ± 0.7

6.1 ± 0.4

SE

23.3 ± 3.3

7.2 ± 1.2

18.4 ± 2.0

19.2 ± 0.6

27.8 ± 1.4

4.2 ± 0.3

1.1 ± 0.4

5.1 ± 1.8

PL

24.2 ± 1.0

3.4 ± 1.1

10.0 ± 1.9

9.8 ± 1.1

29.8 ± 1.0

13.4 ± 0.6

1.9 ± 0.8

7.4 ± 0.8

D E

T P E

U N

SC

A M

C C

TL, total lipid; TAG, triacylglycerol; FFA, free fatty acids; SE, sterol ester; PL, phospholipid

A

29

ACCEPTED MANUSCRIPT

Reengineering lipid biosynthetic pathways of Aspergillus oryzae for enhanced production of -linolenic acid and dihomo--linolenic acid Sukanya Jeennor1  Jutamas Anantayanon1  Sarocha Panchanawaporn1  Sakda

RI

PT

Khoomrung2,3  Chanikul Chutrakul1  Kobkul Laoteng1

Highlights

SC

: -Linolenic and dihomo--linolenic acids production by oleaginous fungus

NU

: Engineering of fatty acid and triacylglycerol biosynthesis in Aspergillus oryzae : The production yields of GLA and DGLA was enhanced by physiological control

AC

CE

PT E

D

MA

: Co-regulation of DGAT2 gene expressions in TAG biosynthesis of the engineered strain

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5