Strains and media
The parent strain in this study was R. toruloides Δku70, a derivative of R. toruloides IFO0880 with a deletion in the non-homologous end joining (NHEJ) gene, ku70 [29]. Agrobacterium tumefaciens AGL-1 strain was used for transformation experiments [18, 30]. The E. coli DH5α was used for plasmid construction.
R. toruloides strains were routinely grown in liquid or solid Yeast Extract–Peptone–Dextrose (YPD) medium (10 g/L of yeast extract, 20 g/L of peptone, 20 g/L glucose) at 30 °C for strain construction and activation, and in fermentation medium (glucose 60.0 g/L, peptone 15.7 g/L, yeast extract 15.7 g/L) for both shake-flask batch fermentation and bioreactor fed-batch fermentation. E. coli strains were routine grown in Luria-Bertani broth (LB) medium at 37 °C. A. tumefaciens was grown at 28 °C in 2YT medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl). Kanamycin, ampicillin, hygromycin, cefotaxime and rifampicin were supplemented to the medium to a final concentration of 50 µg/mL, 100 µg/mL, 100 µg/mL, 300 µg/mL, and 25 µg/mL, respectively. Strain information used in this study are listed in Table 1.
Table 1
Strains and Plasmids used in this study
Strains or plasmids | Relevant characteristics | Source or Reference |
Strains | | |
Δku70 | R. toruloides IFO0880 deficient in ku70 | [31] |
Δku70-AbWS | Δku70 strain harboring the AbWS cassette | This study |
Δku70-AbWS* | Δku70 strain harboring the AbWS* cassette | This study |
Δku70-MmWS | Δku70 strain harboring the MmWS cassette | This study |
Δku70-MhWS | Δku70 strain harboring the MhWS cassette | This study |
Δku70-PaWS | Δku70 strain harboring the PaWS cassette | This study |
Δku70-RoWS | Δku70 strain harboring the RoWS cassette | This study |
AGL-1 | A. tumefaciens strain AGL0 recA::bla pTiBo542ΔT Mop+ CbR | [16] |
DH5α | E. coli (supE44 lacU169 hsdR17 recA1 endA1 gyrA96 thi-l relA1) | Laboratory storage |
Plasmids | | |
pKOCAR2 | Harboring the hygromycin selection cassette for target gene incorporation at the specific site in CAR2 gene | [29] |
p101 | Harboring the MhWS cassette | This study |
p102 | Harboring the AbWS cassette | This study |
p103 | Harboring the RoWS cassette | This study |
p104 | Harboring the MmWS cassette | This study |
p105 | Harboring the PaWS cassette | This study |
p1022 | Harboring the AbWS* cassette | This study |
Plasmid Construction And Yeast Transformation
Standard genetic manipulations were performed as previously described [32]. pKOCAR2 plasmid was used as the gene expression vector for yeast transformation [29]. The WS genes were codon optimized and synthesized by Nanjing GeneScript Biotech Co., Ltd (Nanjing, China). They were amplified and ligated to the pKOCAR2 plasmid using the NEBuiler HiFi Assembly kit (New England Biolabs, USA), yielding plasmids p101, p102, p103, p104, and p105, respectively. The optimized gene sequences and primers used in this study were listed in Tables S1 and S2, respectively.
For enzyme engineering of AbWS, overlap-extension PCR was used for site-directed mutagenesis [33]. Two pairs of primer were designed to amplify the upstream and downstream DNA fragments, respectively. The modified nucleic base was contained in the primers. The mutated AbWS gene was ligated to the pKOCAR2 plasmid, yielding recombinant plasmid p1022. All plasmids used in this study were listed in Table 1.
The recombinant plasmids were transformed into R. toruloides by ATMT as described previously [34]. The pKOCAR2-derived plasmids were firstly electroporated into A. tumefaciens AGL-1. Then the A. tumefaciens strain harboring the corresponding plasmid was co-cultured with the R. toruloides Δku70, and the mixture was spread on YPD solid plate supplemented with hygromycin and cefotaxime for colony screening. Colonies appeared after 2 days.
Fermentation Conditions
Shake-flask batch fermentation was conducted in a 50-ml Erlenmeyer flask containing 10 ml of fermentation medium. The culture was inoculated with 10% seed culture, and performed in an orbital shaker at a rotary rate of 250 rpm at 30 °C. After culturing for 24 h, exogenous ethanol was added to the culture to a final concentration of 1%, 3%, 5%, 7%, respectively. To facilitate the extraction process, 10% dodecane was added to the culture as reported previously [16]. Heptadecanoic acid ethyl ester was used as the internal standard.
Fed-batch fermentation was conducted in a 1-liter stirred bioreactor (Eppendorf, Germany). Culture pH was monitored in real-time by a pH meter (Mettler-Toledo, Switzerland), and dissolved oxygen was monitored in real-time by an oxygen probe (Mettler-Toledo, Switzerland). The initial volume was 500 mL. The cultivation conditions were the same with what was previously described [35]. The glucose and ethanol concentrations were determined by High-Performance Liquid Chromatography (HPLC). The glucose concentration was kept above 10 g/L by feeding 50 mL glucose with a concentration of 500 g/L. 50 mL 50% ethanol was pumped to the fermenter at a flow rate of 1 mL/min to a final concentration of 5% after inoculation of 24 hours. 50 mL dodecane together with ethyl heptadecanoate (C17:0) internal standard were injected to the fermenter through a syringe.
Analytical Methods
The growth curves of the R. toruloides strains were determined by measuring the cell density at 600 nm using a Genesys 20 spectrophotometer (Thermo Fischer Scientific Inc., USA). The dry cell weight (DCW) was determined by the previously described method [5]. The concentrations of glucose and ethanol were monitored by a LC-20A HPLC instrument (Shimadzu Inc., Japan) equipped with a RID detector as previously reported [16].
Total Lipids And Faees Extraction And Analysis
For extracellular FAEEs, the concentration was determined by directly injecting the dodecane layer to a SHIMADZU Japan GCMS-QP2010 PLUS mass spectrometer coupled with a SHIMADZU gas chromatograph (Shimadzu Inc., Japan). The heptadecanoic acid ethyl ester was added to the medium as the internal standard. The FAEEs dissolved in the dodecane layer were separated using a SHIMADZU 5MS capillary column (30 m × 0.25 mm I. D., 0.25 µm film thickness) as previously described [36].
For intracellular FAEEs, the concentration was measured by the following procedure. First, the total lipids were extracted. Cells were harvested and washed twice with double-distilled water (ddH2O), and then freeze-dried until the samples were dry. Lipids were extracted from the lyophilized cells as previously reported [37]. Heptadecanoic acid ethyl ester was added as an internal standard. Second, lipids were separated and fractionated. FAEE components in the total lipid extracts were separated by thin layer chromatography using TLC Silica gel 60 F254 plates as previously reported [13] (Merck, Darmstadt, Germany). The FAEE components were then scraped from the TLC plate and extracted by an organic solvent containing 3 mL hexane, 2 mL methanol and 2 mL ddH2O and vigorously vortexed for 1 h at room temperature. After centrifuging at 3000 rpm for 5 min, the organic phase was transferred to a new glass tube and dried under nitrogen. The residues were dissolved in 200 µL methanol: chloroform (95: 5) for GC-MS analysis.
To determine the fatty acid composition, total lipids were transmethylated and analyzed according to a previously reported method [38].
Western Blot
Western blot
The cells were sampled in the exponential growth period, and homogenized using a FastPrep instrument (MP biomedicals, Solon, OH, USA) for 6 cycles at a speed of 6.0 m/s for 30 s each, with 5 minutes of interval. The suspensions were centrifuged at 15, 000 g for 30 min, and the supernatants were collected [31]. Then the total proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12% polyacrylamide gels, which were then immediately transferred to a polyvinylidene difluoride (PVDF) membrane (BIO-RAD, USA). A Flag tag was added in the 5’ end of the AbWS gene. A monoclonal anti-FLAG M2 antibody was used for Western blot analysis as previously described [39], and the membrane was covered with electrochemiluminescence (ECL) plus solution for color reaction. Finally, the membrane was exposed to the Gel Imager System (Azure, USA) by the chemiluminescence mode.