General Protocol

General Protocol

Experiment Protocol

Experiment Protocol

Aim

The aim of this experiment is to assess the impact of the introduced genetic circuit of PUFA synthase on the lipid profile, especially the DHA accumulation, in engineered Y. lipolytica using Gas Chromatography-Mass Spectrometry (GC-MS). The lipid profile of the non-engineered Y. lipolytica will be used as a control for measuring the changes in the lipid profile in the engineered Y. lipolytica.

Experimental Design

Lipid extraction:

* All centrifugation was performed using Eppendorf Centrifuge 5702 R

• Inoculate a colony of Y. lipolytica from a YPD plate in 15 mL of YPD medium for 1 day (for 24-h culture) or 2 days (for 48-h culture) before the experiment date.
• Harvest 15 mL Y. lipolytica culture by centrifugation at 4,000 x g for 10 min at room temperature. Discard the supernatant.
• Wash the cells with double-distilled H₂O or TE buffer twice.
• Add 4 mL chloroform, 8 mL methanol, and 3.2 mL of 1% NaCl to the pellet, vortex after every addition.
• Agitate on a tube rotator at 30 rpm overnight.
• Add 4 mL chloroform and 4 mL of 1% NaCl, invert 30 times.
• Centrifuge at 4,000 x g for 5 min, and collect the bottom layer.
• Blow dry with a gentle stream of nitrogen.
• (After gravimetric analysis,) Dissolve the lipid in 1 mL of methanol and chloroform mixture (2:1, v/v) to prevent DHA oxidation.
• Samples are stored at or below 4℃ until ready for extraction to minimize oxidation and degradation of samples.

Gravimetric analysis:

• Put an empty beaker in a 90℃ oven overnight and record the weight.
• Put the same empty beaker in the 90℃ oven overnight again and record the weight.
• If the difference between the weights is ±0.0005, acceptable. Take the average of the two values.
• Insert the entire sample in a beaker and blow-dry with nitrogen gas until it is almost dry.
• Put the beaker in the 90℃ oven overnight and record the weight.
• Put the same beaker in the 90℃ oven overnight again and record the weight.
• If the difference between the weights is ±0.0005, acceptable. Take the average of the two values.
• Calculate the difference between the average values from the empty beaker trial and the sample-containing beaker trial. This difference will be the amount of the lipid in the sample.
• Only proceed to subsequent steps if the difference in weight is significant.

FAME (Fatty Acid Methyl Ester):

• 2 mg of the lipid sample are reconstituted in 1 mL of 0.5 M methanolic NaOH and hydrolyzed at 80℃ for 40 min.
• Add 1 mL of fresh 10% BF₃ (dissolved in methanol). Transesterification is performed at 80℃ for 40 min.
• Keep the temperature the same all the time.
• After transesterification, 2 mL of double-distilled H₂O and 1 mL of hexane are added to the sample to quench the reaction.

Gas Chromatography-Mass Spectrometry (GC-MS):

• Sample preparation for testing GC-MS
• Prepare the vials containing the transesterified and quenched lipid samples.
• Prepare the vials containing the standard fatty acids.
• Lipid samples are measured with GC-MS (Thermo Scientific ISQ with TRACE 1300 GC-MS) to confirm peaks.

Results

GC-MS for non-engineered Y. lipolytica:

• For the experiment, three fatty acid standards that are known to be abundant in Y. lipolytica were prepared, namely palmitic acid(C16:0), oleic acid(C18:1), and linoleic acid (C18:2). The GC-MS result of the oleic acid standard was omitted from the report since it was not significantly detected due to random error. Since all conditions for the three standards were the same, this error was unexpected and unable to be explained. Further replication trials for the oleic acid standard would be conducted. If the results from the additional trials are unclear, an oleic acid standard from a different manufacturer would be used.

Figure 1. GC-MS result of Linoleic acid (C18:2) Spectrum of the transesterified linoleic acid (C18:2) sample in GC-MS. The peak of the linoleic acid standard sample is indicated as a single intense peak.

Figure 2. NIST library of linoleic acid As one of the standards, a linoleic acid sample after FAME (Fatty Acid Methyl Ester) was tested. Mass spectra of the peaks were matched against the NIST library.

Figure 3. GC-MS result of Palmitic acid (c16:0)Spectrum of the transesterified palmitic acid (C16:0) sample in GC-MS. The peak of the palmitic acid standard sample is indicated as a single intense peak.

Figure 4. NIST library of palmitic acid As another standard, a palmitic acid sample after FAME (Fatty Acid Methyl Ester) was also tested. Mass spectra of the peaks were matched against the NIST library.

Figure 5. GC-MS result of lipid from Y. lipolytica cultured in YPD medium for 24h

Figure 6. GC-MS result of lipid from Y. lipolytica cultured in YPD medium for 48h

• The GC-MS chromatograms of two standards: palmitic acid (C16:0) and linoleic acid (C18:2), were recognized by comparing their mass spectra to the corresponding references reported in the NIST library (Fig. 1,2,3,4). The presence of chlorine in the linoleic acid sample is hypothesized as the result of chloroform usage during the lipid extraction process. (Fig. 2)
• In the lipid extract from Y. lipolytica samples that were grown in YPD medium for 24 hours and 48 hours, palmitic acid was detected.
• Based on the result, the major lipid component present in non-engineered Y. lipolytica was palmitic acid (C16:0), but linoleic acid (18:2) was not detected.
• The major limitation of this experiment is that the profile of the lipid extracted from Y.lipolytica is unclear.
• Several aspects could potentially be improved:
• First, there were some random errors during the process of lipid extraction before GC-MS analysis, despite the consistent conditions. Certain amounts of lipid pellets might have been accidentally removed together while removing the aqueous layer of the centrifuged samples.
• Second, there were some limitations in the culture medium of Y. lipolytica, so the lipid accumulation in Y. lipolytica was not sufficient.
• In order to measure the amount of lipid extracted from Y. lipolytica, another experiment of gravimetric analysis was conducted between lipid extraction and FAME to check if the lipid samples extracted from the non-engineered Y. lipolytica contained a sufficient amount of lipid.

Lipid amount - gravimetric analysis:

Table 1. OD₆₀₀ measurement and gravimetric analysis result

Figure 7. Lipid extract yield that is normalized to cell weight. The two-tailed P value was 0.0493, considered to be statistically significant.

• Since unknown bacteria were observed in culture with YNB, ampicillin was added to the YNB medium to prevent undesired bacteria growth.
• Considering the potential impact of ampicillin on the growth of the Y. lipolytica, this result should be confirmed again with YNB medium without ampicillin.
• Gravimetric analysis was used to measure weight of the lipid extracted from Y. lipolytica.
• Result shows that YPD has higher OD₆₀₀, mean cell weight, and mean lipid weight. However, their lipid weight normalized by cell weight was lower than that of YNB with ampicillin (Table 1, Fig. 7).
• Hence, it is concluded that YNB is a more advantageous candidate as a growth medium for lipid accumulation in Y. lipolytica.

Future Plan

GC-MS for non-engineered Y. lipolytica (grown in YNB medium)

• Based on the results to date, we observed that Y. lipolytica grown in YPD medium yielded unclear results, while gravimetric analysis indicated that Y. lipolytica grown in YNB medium demonstrated more effective lipid accumulation.
• Therefore, a repeat experiment using Y. lipolytica grown in YNB medium will be conducted, as this may enhance lipid accumulation and provide a clearer control lipid profile.
• A new GC-MS for oleic acid, one of the standards, will also be conducted.

GC-MS for engineered Y. lipolytica (specifically measure the DHA amount)

• Standard: Pure DHA
• Add a known quantity of DHA
• Blank: Lipid from non-engineered Y. lipolytica
• Spiking: Lipid from engineered Y. lipolytica + DHA
• Add a known quantity of DHA
• Target: Lipid from engineered Y. lipolytica
• Observation of the recovery from the spike
• Compare the peak area of the spiked DHA to a standard.
• Calculate the recovery rate using the formula:
• Recovery rate (%) = (Amount from spiking - Amount from target)/(Amount spiked) * 100%
• A recovery rate of 90 to 110% is considered an excellent result.
• If recovery rate falls out of that range, other factors are affecting the result.
• < 90%:
• Recovery less than 90% could be due to the degradation of DHA by β-oxidation enzymes (e.g., acyl-CoA oxidase) (Liu et al., 2021).
• Heat-inactivate the enzymes at above 70℃.
• DHA might have been degraded due to oxidation by air.
• Avoid storage of the samples in the open area to minimize exposure to the air.
• > 110%:
• Recovery over 110% suggests interference by contaminants. Additional DHA can be introduced by contaminations from chemical processing of the samples, or external sources such as manufacturing facilities or laboratory environments.
• Expected results: compared to non-engineered Y. lipolytica, engineered Y. lipolytica has higher DHA amount (approximately 30%).

References

Applegate, B. L. (2007). “Extraction, Derivatization, and Analysis of Fatty Acid Methyl Ester (FAME) in Tissue Homogenates and Blubber by ASE and Gas Chromatography.” Short description of FAME Analysis. Applied Science, Engineering, and Technology Laboratory, University of Alaska Anchorage.
Gemperlein, K., Dietrich, D., Kohlstedt, M., Zipf, G., Bernauer, H. S., Wittmann, C., Wenzel, S. C., & Müller, R. (2019). Polyunsaturated fatty acid production by Yarrowia lipolytica employing designed myxobacterial PUFA synthases. Nature Communications, 10(1), 4055. https://doi.org/10.1038/s41467-019-12025-8
Liu, H., Song, Y., Fan, X., Wang, C., Lu, X., & Tian, Y. (2021). Yarrowia lipolytica as an oleaginous platform for the production of value-added fatty acid-based bioproducts. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.608662

Aim

The aim of this experiment is to examine the influence of media type and different nitrogen source levels, and a peroxisome inhibitor (3-Amino-1,2,4-triazole(3-AT)) in lipid accumulation by using Nile blue and Nile Red staining assay.

Experimental Design:

Previous research shows that yeasts cultured in YNB (YNB-defined synthetic media) have higher lipid accumulation levels in cells than in YPD media (Gasovic et al, 2023). Additionally, nitrogen limitation can increase lipid accumulation levels. However, it only compares the most commonly used YNB media (with 5g/L ammonium sulfate as the nitrogen source) and YNB completely without the nitrogen source (Pomraning et al, 2016). Since nitrogen source levels may also affect the growth of the yeast, we decided to investigate the lipid accumulation levels under different nitrogen source levels and media types.

Some studies show that inhibiting peroxisome in Y. lipolytica can increase lipid accumulation (Xue et al, 2013). 3-amino-1,2,4-triazole (3-AT) has been proven to inhibit peroxisome by inhibiting the peroxisomal catalase (Ueda et al, 2003). We decided to add 3-AT to YPD media to investigate the lipid accumulation levels under the addition of peroxisome inhibitor.

Nile Blue Staining

• Y. lipolytica culture in different media
  • YPD
  • YNB (with 5g/L ammonium sulfate)
• 96-Well Plate Fluorescence Assay
  • Excitation wavelength: 500 nm
  • Emission wavelength: 625 nm
  • Plate reader: FlexStation 3 Multi-Mode Microplate Reader (6 flashes per second)
  • Plate Design (Nitrogen-limiting):

    Table 2. Plate design for Nile Blue staining of Y. lipolytica cultured in YPD and YNB (with 5 g/L ammonium sulfate)

  • Real plate:

    Figure 8. Real plate for Nile Blue staining of Y. lipolytica cultured in YPD and YNB (with 5 g/L ammonium sulfate)

Nile Red Staining

• Y. lipolytica culture in different media
  • YPD
  • YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)
  • YPD (with 10mM 3-AT, a peroxisome inhibitor)
• 96-Well Plate Fluorescence Assay
  • Excitation wavelength: 485 nm
  • Emission wavelength: 535 nm
  • Plate reader: FlexStation 3 Multi-Mode Microplate Reader (6 flashes per second)
  • Plate Design (nitrogen-limiting):

    Table 3. Plate design for Nile Red staining of Y. lipolytica cultured in YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)

  • Real plate (nitrogen-limiting):

    Figure 9. Real plate for Nile Red staining of Y. lipolytica cultured in YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)

  • Plate Design (peroxisome inhibitor):

    Table 4. Plate design for Nile Red staining of Y. lipolytica cultured in YPD and YPD (with 10mM 3-AT, a peroxisome inhibitor)

  • Real plate (peroxisome inhibitor):

    Figure 10. Real plate for Nile Red staining of Y. lipolytica cultured in YPD and YPD (with 10mM 3-AT, a peroxisome inhibitor)

Results:

Expected Results:

• The fluorescence of Y. lipolytica culture in YNB media is higher than in YPD media, indicating higher lipid accumulation levels.
• The fluorescence of Y. lipolytica culture in YNB with lower nitrogen source levels is higher than in YNB with higher nitrogen source levels, indicating higher lipid accumulation levels.
• The fluorescence of Y. lipolytica culture in YPD with 3-AT is higher than in YPD without 3-AT, indicating higher lipid accumulation levels.

Nile Blue Staining

• The average fluorescence of Y. lipolytica cultured in YNB is 1.814, while 0.152 in YPD.
• Y. lipolytica cultures in YNB media have higher lipid accumulation levels than in YPD media.

Figure 11. Nile Blue staining result of Y. lipolytica cultured in YPD and YNB (with 5 g/L ammonium sulfate)

Nile Red Staining of nitrogen limiting

• The differences in fluorescence between Y. lipolytica cultures in YNB media with different nitrogen source levels are minor and fluctuant, especially in the range of 1 g/L to 10 g/L.
• Based on the results, nitrogen limitation may not lead to significant changes in lipid accumulation levels in Y. lipolytica.

Figure 12. Nile Red staining result of Y. lipolytica cultured in YNB (with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g/L ammonium sulfate, respectively)

Nile Red Staining of peroxisome inhibitor

• Y. lipolytica cultures in YPD media (with 10mM 3-AT, a peroxisome inhibitor) have higher lipid accumulation levels than in YPD media.
• Inhibition of peroxisome in Y. lipolytica can lead to increased lipid accumulation levels.

Figure 13. Nile Red staining result of Y. lipolytica cultured in YPD and YPD (with 10mM 3-AT, a peroxisome inhibitor)

References

Jovanovic Gasovic, S., Dietrich, D., Gläser, L., Cao, P., Kohlstedt, M., & Wittmann, C. (2023). Multi-omics view of recombinant Yarrowia lipolytica: Enhanced ketogenic amino acid catabolism increases polyketide-synthase-driven docosahexaenoic production to high selectivity at the gram scale. Metabolic Engineering, 80, 45–65. https://doi.org/10.1016/j.ymben.2023.09.003
Pomraning, K. R., Kim, Y.-M., Nicora, C. D., Chu, R. K., Bredeweg, E. L., Purvine, S. O., Hu, D., Metz, T. O., & Baker, S. E. (2016). Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica. BMC Genomics, 17(1), 138. https://doi.org/10.1186/s12864-016-2471-2
Rakicka, M., Lazar, Z., Dulermo, T., Fickers, P., & Nicaud, J. M. (2015). Lipid production by the oleaginous yeast Yarrowia lipolytica using industrial by-products under different culture conditions. Biotechnology for Biofuels, 8(1), 104. https://doi.org/10.1186/s13068-015-0286-z
Rostron, K. A., & Lawrence, C. L. (2017). Nile red staining of neutral lipids in yeast. In C. Pellicciari & M. Biggiogera (Eds.), Histochemistry of Single Molecules (Vol. 1560, pp. 219–229). Springer New York. https://doi.org/10.1007/978-1-4939-6788-9_16
Ueda, M., Kinoshita, H., Yoshida, T., Kamasawa, N., Osumi, M., & Tanaka, A. (2003). Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo. FEMS Microbiology Letters, 219(1), 93–98. https://doi.org/10.1016/S0378-1097(02)01201-6
Xue, Z., Sharpe, P. L., Hong, S.-P., Yadav, N. S., Xie, D., Short, D. R., Damude, H. G., Rupert, R. A., Seip, J. E., Wang, J., Pollak, D. W., Bostick, M. W., Bosak, M. D., Macool, D. J., Hollerbach, D. H., Zhang, H., Arcilla, D. M., Bledsoe, S. A., Croker, K., … Zhu, Q. (2013). Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nature Biotechnology, 31(8), 734–740. https://doi.org/10.1038/nbt.2622

Acknowledgement

We thank Biosciences Central Research Facility of HKUST (Clear Water Bay) to support the work in general equipment core.

Aim

The aim of this experiment is to examine the concentration of DHA in engineered Y. lipolytica culture.

Experimental Design

DHA standard curve:

• DHA has a specific absorbance peak at 205nm, which can be detected by spectrophotometers. Those absorbance values are linearly correlated to DHA concentration. Hence, a DHA standard curve was measured in order to examine the concentration of DHA in yeast culture.
• Various concentrations (1, 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500 μg/mL) of the DHA sample were prepared, using absolute ethanol as solvent.
• The spectrum of pure DHA samples within 190-300nm was analyzed by spectrophotometer.
• A standard curve was plotted using various concentrations measured at 205nm wavelength.

* All measurement was performed using BioDrop μLite Spectrophotometer.

Results

• A linear curve of DHA concentration and 205nm absorbance.
• Equation: y = 0.0044x + 0.0791, R²= 0.9852

Figure 14. DHA Standard Curve (205nm)

Future Plan

DHA measurement:

• Extract lipid from engineered Y. lipolytica culture and dissolve in absolute ethanol.
• The 205nm wavelength of the sample will be measured by a spectrophotometer.

* All measurement was performed using BioDrop μLite Spectrophotometer.

• The DHA concentration of the sample will be calculated using the equation y = 0.0044x + 0.0791.
• If the absorbance of the sample solution is out of range (1-500 μg/mL), dilute the sample or use less absolute ethanol to dissolve the lipid.
• Expected result: The DHA production of engineered Y. lipolytica culture is around 3g/L or more.

Reference

Sahi, A. K., Anjali, Varshney, N., Poddar, S., Vajanthri, K. Y., & Mahto, S. K. (2019). Optimizing a detection method for estimating polyunsaturated fatty acid in human milk based on colorimetric sensors. Materials Science for Energy Technologies, 2(3), 624–628. https://doi.org/10.1016/j.mset.2019.07.001

Aim

The aim of this experiment is to examine the growth of Y. lipolytica in medium with glucose versus glycerol as the carbon source. The recorded data is analysed to estimate the time period of each growth phase, the carrying capacity, and the theoretical growth rate.

Experimental Design

Y. Lipolytica Growth Curve:

• Yeast Y. lipolytica cell growth in liquid medium can be monitored using OD600nm.
• Cell growth is monitored at certain time points after inoculation, an spectrophotometer is used to measure its OD600 value.
• The measurements are plotted against time elapsed after inoculation, and the data is fitted to a logistic curve.
• The growth curves are compared to identify the optimal carbon source.

Conditions

General Conditions:

• 10g/L Yeast Extract
• 20g/L Peptone
• 120mL media in 1000mL conical flask
• Shaking Incubator: 30˚C ± 2; 225rpm
• Initial pH: N/A
• Initial inoculum: 2 night Y. lipolytica in YPD, washed twice with PBS
  • Final resuspension in sterile water, add 1mL to each flask
  • Same amount is added to each flask

Variable Conditions:

• 20g/L Glucose (Control)
• 20g/L Glycerol
• 40g/L Glucose
• 40g/L Glycerol

Results & Analysis:

Fit to Logistic Curve
• Graph

Figure 15. Sept 16th Growth Curve

First Derivative :

• Estimated Maximum Growth Rate:

  Steps	Function
  Carbon Source	Maximum Growth Rate
  20g/L Glucose (Control)	0.2245
  20g/L Glycerol	0.2410
  40g/L Glucose	0.1840
  40g/L Glycerol	0.2725

• Graph:

Figure 16. Sept 16th First Derivative

Results

From our results, we identified that glycerol has a higher maximum growth rate than glucose for both concentrations. It was also observed that the cell culture reaches mid exponential phase earlier when using glycerol as the carbon source, which agrees with our modelling results.

Future Plan

• Repeating the Experiment
• Testing Waste Carbon Sources:

Y. lipolytica is known of its ability to utilize waste carbon sources for growth, such as crude glycerol and waste cooking oils. In the future, we hope to evaluate the efficiency of using waste carbon sources as media to reduce the environmental impact and costs of yeast cultivation, as well as potential future work to further optimize our production process.

Aim

The aim of this experiment is to assess the expression of each subunit of PUFA synthase.

Experimental Design

Each subunit of PUFA synthase is attached to a His-tag, so we use a His-tag antibody as our primary antibody. An anti-Rabbit/Mouse IgG antibody is used as our secondary antibody.

Protein Sample Preparation

• Harvest Y. lipolytica and lyse the cells.
• Extract proteins from Y. lipolytica cells.

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

• Set up and run a polyacrylamide gel.

Western Blot

• Use anti-His-tag primary antibody and appropriate secondary antibody to detect the subunit of PUFA synthase.

Expected Results

We will be able to detect the subunit of PUFA synthase in our engineered Y. lipolytica.

Reference

Nevada. (2014). Western Blot to Examine Yeast Strains for Protein Expression. iGEM. https://static.igem.org/mediawiki/2014/5/52/UNR_Western_Blot_Protocol_to_detect_GFP_in_yeast.pdf