Fungal strain and growth conditions
Aspergillus carbonarius ITEM 5010 (ATCC® MYA-4641™) was used as the parent strain in all the transformations. The strains were grown on Potato Dextrose Agar for 5 days at 30 °C for preparation of conidial suspensions. Sterile double distilled water was added to the plates to collect conidia from the surface of the agar, and the suspension was filtered through sterile Miracloth (Millipore, Billerica MA, USA) and counted in a haemocytometer. Transformants were maintained on minimal medium agar (Samson et al. 2004) supplemented with 100 µg/ml Hygromycin B. Alkane production was studied on glucose (composition same as minimal medium), oatmeal and Yeast Malt medium (YM, 3% yeast extract, 3% malt extract, 10% glucose). Glucose was substituted with oatmeal (20 g/l) for the oatmeal based medium. All chemicals were purchased from Fisher Biotech (Fair lawn, NJ, USA), unless otherwise stated.
Gene description and codon optimization
Synechococcus elongatus strain PCC7942 (ATCC® 33912™) aldehyde deformylating oxygenase (Synpcc7942_1593) and acyl-ACP reductase (Synpcc7942_1594) were the two genes used in this study. Codon optimization and gene synthesis were carried out by Genscript USA Inc. (Piscataway, NJ, USA). The optimization was carried out based on the codon usage frequency of the closely related strain Aspergillus niger (Abarca et al. 2004). The sequence of the codon optimized FAR and FADO genes can be retrieved with Genbank accession numbers KX903286 and KX903287, respectively.
Plasmids and expression vectors
The promoters of genes encoding Tef1 and CoxA, from Aspergilus nidulans and A. niger, respectively, and the terminators of genes TrpC and CoxA also originating from A. nidulans and A. niger, respectively, were used for the heterologous expression of the codon optimized genes in A. carbonarius. The promoters and terminators were PCR amplified from the genomic DNA of their parent strains (kindly donated by Pacific Northwest National Laboratory, Richland, WA). The sequences of oligonucleotides used in this study are provided in Additional file 1: Table S1.
The FAR/FADO expression vector consisted of the codon optimized FAR flanked by Tef1 promoter and CoxA terminator and the codon optimized FADO with CoxA promoter and TrpC terminator. The promoters, genes and terminators were cloned into plasmid pCB1004 (Fungal Genetics Stock Center, Manhattan, KS, USA), which contains Hygromycin B fungal selection marker, by Gibson Assembly Cloning (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s protocol. The resulting expression vector, pCB1004TDR (Additional file 1: Figure S1), was used to transform protoplasts of the parent strain.
Transformation of A. carbonarius ITEM5010
Ectopic integration of the expression vector into the genome of A. carbonarius ITEM 5010 was achieved via protoplast transformation. Protoplast preparation and transformation was carried out as previously described by Gallo et al. (2014). The transformant heterologously expressing the FAR and FADO genes is referred to as TDR transformant.
Selection of transformants, DNA extraction, and expression analysis
Randomly selected transformants were isolated on selective minimal medium agar. Genetically stable homokaryons were achieved by three successive rounds of plating conidia of the selected transformants on selective medium and isolation of single colonies. Genomic DNA (gDNA) of transformants was isolated by the Cetyl trimethylammonium bromide (CTAB) and phenol–chloroform extraction method, previously described by Lee et al. (1988) and was used as template for the PCR verification of the transformants for the presence of the correct inserts, using oligonucleotides IW501 and IW512 (Additional file 1: Table S1) and Phusion polymerase (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s protocol. Based on the PCR verification, seven transformants were selected for further analysis for expression of the inserted genes. Total RNA was extracted by following the manufacturer’s protocol of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Complementary DNA (cDNA) was synthesized from mRNA by using the Verso cDNA Synthesis Kit (Thermo Scientific, Pittsburgh, PA, USA), according to the manufacturer’s protocol and verified by PCR using oligonucleotides presented in Additional file 1: Table S1. The PCR verification consisted of the amplification of a few hundred base pairs of the terminal region of both the FAR and FADO genes from the selected transformants. Beta-actin was used as control to confirm that cDNA had been synthesized and that the samples did not contain traces of genomic DNA that would interfere with proper assessment of the PCR results. Amplification of a short terminal region of beta-actin yields fragments of different sizes from gDNA and cDNA due to the presence of an intron.
Southern blot analysis
The hybridization probe for the southern blot analysis of the TDR transformants was designed to consist of a single fragment containing FADO, TprC terminator, tef1 promoter and FAR sequence, originating from the previously constructed expression vector (Fig. 3a). The DNA probe was biotin labeled by using Pierce North2South Biotin Random Prime Kit (Thermo Fisher Scientific, Rockford, IL, USA) according the manufacturer’s protocol. Genomic DNA of the transformants was extracted as described above and digested with restriction enzymes BamHI, PstI, and NdeI (all purchased from Thermo Scientific, Rockford, IL, USA). Southern blotting was carried out using Whatman Turboblotter transfer system (GE Healthcare Life Sciences, Pittsburg, PA, USA) and the nucleic acid detection was carried out by using Pierce Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, Rockford, IL, USA), both according to the manufacturer’s protocol.
Flask fermentation of the selected transformant and the parent strain were carried out using three different carbon based media: glucose, oatmeal, and YM medium. All three media were supplemented with 1% Tween 80. Each flask (500 ml, non-baffled) contained 100 ml medium which was inoculated with fungal conidia to a final concentration of 5 × 105 conidia/ml. The cultures were grown on 30 °C at 140 rpm for 6 days in a shaking incubator. All fermentations were carried out in triplicates, and a control flask was run parallel without any fungal inoculum.
Internal free fatty acid and triglyceride assay
Lyophilized hyphae of the enhanced expression strain and the parent strain, cultured on the glucose medium, were prepared for analysis using a method previously described by Tamano et al. (2013). The analysis for free fatty acid and triglyceride concentrations was carried out using a commercial free fatty acid kit (Free fatty acids, Half-micro test kit; Roche Applied Science, Mannheim, Germany) and a triglyceride kit (Triglyceride colorimetric assay kit; Cayman Chemical Company, Ann Arbor, MI, USA) following the manufacturer’s protocol.
Fatty acid methyl ester (FAMEs) analysis
Fatty acid methyl ester reaction of the samples was carried out following a method developed by O’Fallon et al. (2007) with lyophilized hyphae (here and below DCW) of the TDR transformant and the parent strain, originating from the culturing on glucose medium, using tridecanoic acid as internal standard. The FAMEs were detected by using gas chromatography (GC)/Flame ionization detector (FID) GC-system model # 6890 N (G1540 N), Agilent Technologies, Wilmington, DE, USA, equipped with a DB-WAX column (30 m × 0.53 mm × 1.00 µm, Agilent Technologies, Wilmington, DE, USA). Retention times of the detected peaks were compared to authentic standards (FAME mix C8-C22, Sigma-Aldrich, St. Louis, MO, USA).
GC/MS sample preparation and alkane analysis
After fermentation, the cultures were filtered through Miracloth (Millipore, Billerica MA, USA). Twenty milliliters of each filtrate was transferred to 50 ml glass centrifuge tubes. Ten milliliters of hexane was added to all tubes, followed by ultra-sonication for 60 min at room temperature, and then vortex for 5 min at maximum speed. Finally, the tubes were centrifuged at 2800g for 10 min and the upper hexane layer was analyzed by gas chromatography/mass spectrophotometry (GC/MS) (7890A GC-system with 5975C inert XL E1/C1 MSD model # G3174A, Agilent Technologies, Wilmington, DE, USA). The samples were analyzed on DB-5MS, non-polar (30 m × 0.250 μm × 0.25 × μm) column, using the following method: 1 μl splitless injection (inlet temperature held at 300 °C) onto the column, the oven was held at 30 °C for 1 min. The temperature was ramped up to 200 °C by 10 °C/min and was held at 200 °C for an additional 1 min. The flow rate of the carrier gas helium was 20 ml/min. Retention times of product peaks were compared with authentic standards (Sigma Aldrich, St. Louis, MO, USA) to confirm peak identity. The quantification of the compounds was done by the external standard method (Zhang et al. 2007, 2008). Three commercial alkane standards (Sigma Aldrich, St. Louis, MO, USA) were prepared at 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l (ppm) concentrations in hexane. Pentadecane and heptadecane standards were used to quantify the alkanes. Tridecane standard was used as internal standard. For accurate quantification purposes, undiluted and 10 times diluted samples were run.