Covering the Land of Lincoln

Manipulation of sterol homeostasis for the production of 24-epi-ergosterol in industrial yeast

Chemicals and reagents

All chemicals and kits were purchased from Sangon Biotech (Shanghai, China) unless specifically mentioned. The ergosterol standard, polyethylene glycol (PEG), and deoxyribonucleic acid sodium salt from salmon testes (single-stranded DNA, ssDNA) were purchased from Sigma-Aldrich. PrimeStar DNA polymerase, Ex Taq® DNA Polymerase, and all restriction enzymes were purchased from Takara (Dalian, China). Phanta® Max Super-Fidelity DNA Polymerase and ClonExpress II One Step Cloning Kit were purchased from Vazyme (Nanjing, China). Yeast Plasmid Extraction Kit and DNA Gel Purification Kit were purchased from Solarbio Life Science (Beijing, China) and Thermo Scientific (Massachusetts, USA), respectively.

Strains and media

Yeast strains used in this study are listed in Supplementary Data 2. Yeast strain S1 (CICC1746) is an industrial strain for ergosterol production and was obtained from the China Center of Industrial Culture Collection. BY4741 is kindly provided by Prof. Zhinan Xu (Zhejiang University, China). CICC1746 genomic DNA was used for the amplification of ARE1, ARE2, YEH1, YEH2, and TGL1. Escherichia coli Trans-T1 (TransGen Biotech, China) was used as the host to construct, maintain, and amplify plasmids. E. coli strains were cultured in LB medium with 50 μg mL−1 ampicillin. Yeast strains were cultivated in YPD medium (1% yeast extract, 2% peptone, and 2% d-glucose), with 100 μg mL−1 hygromycin B (HygB) and 200 μg mL−1 geneticin (G418) supplemented when necessary. YPD medium containing 100 μg mL−1 HygB and 0.025% SDS was used for high-throughput screening of ArDWF1 mutants.

Plasmid construction

All plasmids and primers (synthesized by Sangon Biotech, Shanghai, China) used in this study are listed in Supplementary Data 3 and Supplementary Data 4, respectively. Kits used in DNA manipulation were purchased from Sangon Biotech (Shanghai, China). General DNA amplification from genomic DNA was carried out according to the standard protocol of Phanta® Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). A yeast plasmid extraction kit was purchased from Solarbio Life Science (Beijing, China).

DWF1 genes (AtDWF1, ArDWF1, BrDWF1, and CsDWF1) were codon-optimized for yeast expression and synthesized by Sangon Biotech, which were subsequently cloned into pRS42H, resulting in the construction of the plasmid pRS42H-AtDWF1, pRS42H-ArDWF1, pRS42H-BrDWF1, and pRS42H-CsDWF1, respectively. Plasmid pRS42H-SpCas9 and pKan100-ADE2.136, from Prof. Huimin Zhao (University of Illinois at Urbana‐Champaign, Urbana, Illinois), were used for genome editing in yeast. Specific N20 (from E-CRISP online tool37) of the guide RNA (gRNA) was introduced in the primers used for amplification of the entire pKan100-ADE2.1 plasmid by inverse polymerase chain reaction (PCR)38. The PCR product was then digested by DpnI and transformed into E. coli for amplification.

For co-expression of genes involved in sterol acylation and sterol ester hydrolysis, pEB-3-11 with three expression cassette was constructed based on pEASY®-Blunt Simple Cloning Vectors (TransGen Biotech, Beijing, China). To replace PTEF1 in pRS42H-Ar207 with PTDH3, PERG4, PERG5PCIT2, and PGAL10-PGAL1, the vector scaffold was amplified by reverse PCR using R-pRS42H-207-F and R-pRS42H-207-R, the desired promoters were obtained by PCR from CICC1746 genome, which were recombined to construct pRS42H-PTDH3-Ar207, pRS42H-PERG4-Ar207, pRS42H-PERG5-Ar207, pRS42H-PCIT2-Ar207, and pRS42H-PGAL10-PGAL1-Ar207, respectively.

As for the construction of donor DNAs for the deletion of ERG4, ERG5, ARE1, ARE2, YEH1, YEH2, and TGL1, the upstream and downstream fragments were amplified from the CICC1746 and pieced together using overlap extension PCR. The full-length donor DNA fragments were gel purified and cloned into the pEASY®-Blunt Simple Cloning Vectors (TransGen Biotech, Beijing, China), to create pEASY-erg4Δ, pEASY-erg5Δ, pEASY-are1Δ, pEASY-are2Δ, pEASY-yeh1Δ, pEASY-yeh2Δ, and pEASY-tgl1Δ, respectively. To construct donor DNAs for the integration of ARE1, ARE2, YEH1, YEH2, and TGL1 expression cassettes, the corresponding genes were cloned into pRS42H using ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China).

Yeast transformation and strain construction

CRISPR/Cas9 guided gene knockout and integration were performed with some modifications36. Yeast cells were transformed by the PEG/ssDNA/LiAc method39. The Cas9-expressing strains were constructed by transforming pRS42H-SpCas9 (harboring HygB for hygromycin B resistance) into the corresponding yeast strains. For the co-transformation of gRNA expression plasmids (harboring KanMX for G418 resistance) and donor DNAs into Cas9-expressing strains, heat shock time was prolonged to 50 min, and the yeast strains were recovered in 1 mL YPD for 6 h to allow sufficient expression of the G418 resistance gene. Then the positive transformants harboring pRS42H-SpCas9 and gRNA expression plasmid were selected on YPD/HygB+G418 plates and subsequently confirmed by colony PCR and DNA sequencing.

Directed evolution of DWF1

For the directed evolution of DWF1, the mutant library with two 35 bp homologous arms to PTEF1 and TADH2 in pRS42H was generated by error-prone PCR using Ex Taq® DNA Polymerase (Takara, Dalian, China) with primer pairs of Ep-ArDWF1-F & Ep-ArDWF1-R. The concentration of Mn2+ was set as 0.07 mM. Recombination-mediated PCR-directed plasmid construction in vivo in yeast was performed with some modifications40. Linearized vector and insert fragments from error-prone PCR were co-transformed into YQE101 in a molar ratio of 1:6, and then the transformants were selected on YPD/HygB+0.01% SDS plates. The colonies in the plates were picked and cultured in 2 mL YPD medium containing 100 μg mL−1 HygB as well as 0.025% SDS in 24-well plates. After three days, the grown strains were selected and cultured in 5 mL YPD/HygB to quantify the production of 24-epi-ergosterol. To minimize the effect of HygB on cell growth, the activity of DWF1 was defined as the ratio of the HPLC peak areas of 24-epi-ergosterol and ergosta-5,7,22,24(28)-tetraen-3β-ol. Plasmids of the positive mutants were extracted using a yeast plasmid extraction kit (Solarbio, Beijing, China) and transformed into E. coli. The corresponding mutations were verified by DNA sequencing. To evaluate the contribution of each mutation to improved DWF1 activity, single mutants of DWF1 were constructed by reverse PCR-based site-directed mutagenesis using PrimeStar DNA polymerase (Takara, Dalian, China).

24-epi-ergosterol production in shake flasks and fed-batch bioreactors

Shake-flask experiments were carried out in biological triplicates in 250 mL shake flasks containing 50 mL YPD. Single colonies were inoculated into 5 mL YPD and incubated at 30 °C for 24 h, and then transferred to 250 mL shake flasks with an initial OD600 of 0.1. The yeast strains were cultured at 30 °C and 220 rpm for 96 h.

For fed-batch cultivation, Single colonies were inoculated into 5 mL YPD medium and cultured at 30 °C and 220 rpm for 24 h and then transferred into 250 mL shake flasks containing 50 mL of YPD medium. After 20 h cultivation, yeast cells from two shake flasks were used to inoculate 0.9 L fermentation medium (10 g L−1d-glucose, 10 g L−1 (NH4)2SO4, 8 g L−1 KH2PO4, 3 g L−1 MgSO4, 0.72 g L−1 ZnSO4.7H2O, 10 mL L−1 trace metal solution, and 12 mL L−1 vitamin solution) in a 2 L bioreactor (T&J-MiniBox, Shanghai, China). Fermentations were carried out at 30 °C, and pH was controlled at 5.0 by the automatic addition of 5 M ammonia hydroxide. Dissolved oxygen (DO) was maintained at >25% saturation by adjusting the agitation rate (300 to 950 rpm) and airflow rate (1 vvm to 3 vvm). After the complete consumption of initial glucose and residual ethanol, a feeding solution containing 500 g L−1 glucose and 12 mL L−1 vitamin solution was fed into the bioreactor, based on the pseudo-exponential feeding model developed by O’Connor41. Afterward, to improve the intracellular accumulation of 24-epi-ergosterol, ethanol was fed to the fermenter at a rate of 5 or 6 mL h−1, maintaining DO above 25%. Until the end of the fermentation. The feeding rate FS during the pseudo-exponential feeding phase was determined by the following equations42:

$${F}_{s=}left(frac{mu }{{Y}_{frac{x}{s}}}+mright)cdot frac{{X}_{0}{V}_{0}}{S}cdot {{{{{{rm{e}}}}}}}^{mu t}$$


Where X0, V0, and S were the initial biomass density (gDCW L−1), the initial culture volume (L), and the glucose concentration (g L−1) in the feeding medium; YX/S was the yield of the cell biomass on glucose (gDCW per g glucose); μ was the specific growth rate (h−1); m was the maintenance coefficient (g glucose gDCW−1 h−1), and t was the time (h) after starting the feeding. A predetermined specific growth rate24 of 0.12 h−1 was used to avoid overflow metabolism. The values of YX/S and m were 0.5 and 0.05, respectively43. The feeding rate was adjusted every hour, according to the theoretical model.

Real-time quantitative PCR analysis

Total RNA was isolated from yeast cells by TRIzol (Invitrogen) according to the manufacturer’s instructions. The degradation of genomic DNA and reverse transcription were conducted by ReverTra AceTM qPCR RT Master Mix (TOYOBO, Japan). Real-time Quantitative PCR was performed by 2×T5 Fast qPCR Mix (SYBR Green I) (TSINGKE, China) on LineGene 9600 Plus FQD-96A (Bioer Technology, China). The ACT1 gene (encoding actin) was used as the internal control for expression level normalization. The transcription level was analyzed using the 2−ΔΔCT method44. The primers for ACT1, ARE2, YEH1, YEH2, ACC1, Ar207, and ERG5 were synthesized by TSINGKE, and the corresponding sequences were listed in Supplementary Data 4.

Analytical methods

Cell growth was monitored by measuring optical density at 600 nm (OD600) using a spectrophotometer (721 G, INESA, Shanghai, China). Glucose and ethanol concentrations were determined using a biosensor (SBA-40C; Biology Institution of Shandong Academy of Science, Jinan, China). 24-Epi-ergosterol and precursor sterols were extracted from yeast cells with some modification45. About 500 μL yeast cell culture was harvested and washed twice using ddH2O. About 600 μL alcoholic KOH solution (25% [w/v] in 50% ethanol) was added to the yeast pellets, which were vortexed for 1 min. Cell suspensions were then boiled for 1 h. After cooling on ice, sterols were extracted with 800 μL petroleum ether, followed by a vigorous vortex for 3 min. About 500 μL petroleum ether (top) layer was collected and dried with a vacuum dryer. Dried samples were dissolved in 500 μL ethanol and analyzed by HPLC, equipped with a Thermo C-18 column (ODS Hypersil, 4.6 × 250 mm, 5 μm) and a UV detector at 280 nm. Methanol/acetonitrile (80:20, v/v) was used as the mobile phase with an elution rate of 1 mL min−1. LC-MS was performed through AB Sciex Triple TOF 5600+, equipped with a Thermo C-18 column (ODS Hypersil, 4.6 × 250 mm, 5 μm) and an atmospheric pressure chemical ionization (APCI) ion source. Sterols were separated on methanol/acetonitrile (80:20, v/v) over 20 min with a flow rate of 0.8 mL min−1 at 30 °C. MS was operated in positive ionization mode, with a scanned m/z range of 100–1500. All results were reported as the average of at least three biological replicates.

The analysis of free sterols and sterol esters was performed with some minor modifications46. About 4 mL yeast cell culture was harvested, washed twice using ddH2O, and resuspended in 800 μL TE buffer. Then, 1 g glass beads (425–600 μm, acid washed, Sigma) was used to disrupt the cell walls mechanically by vigorous vortex for 10 min (30 s vortex and 30 s on ice). Afterward, sterols were extracted with 3.2 mL petroleum ether, followed by vigorous vortex for 3 min. For each extraction, a 500 μL petroleum ether (top) layer was collected separately in two tubes and dried with a vacuum dryer. One tube was analyzed by HPLC directly to determine un-acylated late sterols, while the other was subject to a saponification procedure before quantitative analysis by HPLC to determine total late sterols.

Preparation of 24-epi-ergosterol for NMR analysis

About 120 mL yeast cells were collected and resuspended in 240 mL ethanol–KOH solution (KOH: ethanol: H2O = 25:50:50, w/v/v) in a 500 mL shake flask. The reaction mixtures were incubated at 95 °C for 2 h. After cooling to room temperature, the samples were extracted with petroleum ether (boiling point range, 60–90 °C) (3 × 200 mL). The organic layer was dried over Na2SO4 and evaporated, which was further purified by column chromatography using n-hexane:ethyl acetate (100:1) as the eluent. The resultant crude 24-epi-ergosterol sample also included the upstream precursors ergosta-5,7,22,24(28)-tetraen-3β-ol and ergosta-5,7-dien-3β-ol. The crude product was completely dissolved in 5–7 volumes of solvent (n-hexane:ethyl acetate = 1:1) at 60 °C and then cooled down to room temperature for 5 h. The precipitated solid was filtered and the recrystallization procedure was performed for multiple cycles until the purity of 24-epi-ergosterol was sufficient for NMR analysis. The purified 24-epi-ergosterol and ergosterol standard were dissolved in CDCl3 for 500 M NMR analysis (BRUKER DMX-500).

Statistics and reproducibility

All experimental data were at least in triplicate and expressed as mean ± standard error. All data analyses were performed by Excel or OriginPro.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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