Introduction

The aim of this project is to synthesize the important precursor of DHA-PC, DHA-CoA. We utilized protein engineering to modify the wild-type LACS (Long-Chain Acyl-CoA Synthetase). The optimal protein with high affinity for DHA was purified by FPLC (Fast Protein Liquid Chromatography). Afterwards, the NADH couple-enzyme assay [1] was used to compare the different affinities of LACS. In order to detect the product DHA-CoA, we used LC-MS to observe and analyze the peak pattern and area of DHA-CoA. Finally, we got optimized LACS and DHA-CoA generation was detected.

To determine which LACS1 sequence to mutate, we borrowed strain YB525 from Professor Zhu's lab, which had the FAA1 and FAA4 genes knocked out. The aim of this experiment is to quantitatively assess the growth curve of YB525 yeast strain and investigate the substrate affinity of three enzymes (LACS1 BBa_K5255000, Czlacs5 BBa_K5255001, LACS6 BBa_K5255002) towards different fatty acids. Specifically, the study evaluated whether these LACS enzymes demonstrate higher affinity for DHA (22-carbon fatty acid) compared to shorter-chain fatty acids like C16 and C18.

YB525 Growth Curve
Figure 1 | The figure depicts the growth curves of LACS1 (BBa_K5255000), czLACS5 (BBa_K5255001), and LACS6 (BBa_K5255002) following their introduction into strain YB525 via an inducible expression medium augmented with palmitic acid (16:0), stearic acid (18:0), and DHA (22:4) respectively for a 60-hour incubation period.

Preliminary analysis suggests that LACS1 (BBa_K5255000) has a higher affinity for DHA as growth recovery is observed when DHA is supplemented, though not as fast as with C16 and C18.

System in Saccharomyces cerevisiae

Protein Expression in YB525

In order to better express our target protein, we introduced the pYES2 plasmid with the target gene into YB525 and induced expression with 2% galactose for 48 hours. Subsequently, the total protein of YB525 was obtained by pressure crushing to determine the production of the target protein.

Western Blot YB525
Figure 2 | This is a western blot of the induced expression YB525 strain. NC was a negative control, which was the result of uninduced strain pressure crushing. The sample supernatant is the centrifugal supernatant after pressure crushing, and the precipitate is the corresponding precipitate. The whole cell is the protein result before pressure crushing. The results showed that YB525 contained the target protein LACS1 (BBa_K5255000).

Product Detection

Then, the small molecules in the upper clear layer were centrifuged, and DHA-CoA was detected by LC-MS.

LC-MS Detection
Figure 3 | This is the result of the LC-MS assay of the enzyme-catalyzed product. The sample is the supernatant after induced cell breakage, while NC is the supernatant after non-induced cell breakage. An equal amount of internal standard C16-CoA is added to NC and Sample, respectively. The peak area represents the relative amount of the corresponding substance. It can be inferred that DHA-CoA was produced in yeast YB525 after induction.

Protein Engineering in E. coli

Protein Expression and Purification

BL21(DE3) and C43(DE3) were used as chassis to express wild-type and mutant proteins. The protein was harvested 18 hours after induction at 18℃ at 400 μM.

Western Blot BL21
Figure 4 | This is a western blot of the induced expression strain BL21(DE3). "Before" is the sample before ultrasonic cracking, and the first supernatant and the second supernatant are the results of centrifugation after ultrasonic cracking. The results show that LACS1 is mainly an insoluble protein in the precipitation.
Western Blot C43
Figure 5 | This is a western blot of the induced expression of MLACS1 in C43(DE3). The whole cell before ultrasonic lysis, the supernatant, and lower precipitation after ultrasonic lysis were respectively taken for western blot. The results show that there are obvious target bands in the lower precipitation and the size is correct. In addition, there are fewer impurity bands.

In order to obtain the target protein, FPLC was used, and the target protein was purified by nickel ion.

Protein Purification
Figure 6 | Coomassie brilliant blue-stained gel (A) and Western blot (B) of LACS1 proteins purified from BL21(DE3) by fast protein liquid chromatography and separated by SDS-PAGE. Lane 1-2: Samples from the Elution step with the target protein indicated by the arrow (77.7 kDa).

We found that the purified protein expressed in BL21(DE3) had many small bands of miscellaneous proteins, so the protein expressed in BL21(DE3) needed to be purified by anion chromatography.

Anion Chromatography
Figure 7 | This is the result of the protein purified by anion chromatography. The numbers represent the different treatment groups after protein concentration. It shows the relatively high purity of our protein (77.7 kDa) after anion chromatography.

Protein Activity Verification

We used LC-MS to measure the production of DHA-CoA. The catalytic effects of mutant and wild-type enzymes were compared by comparing the amount of product produced in a certain period of time (2 min).

LC-MS Results
Figure 8 | This is the result of testing the product with LC-MS and searching for molecular weight. The blue peaks are the result of the MLACS1 sample, and the red peaks are the result of the LACS1 sample. The peak area represents the relative amount of the corresponding material. The results show that the catalytic activity of the mutant protein (MLACS1) is significantly higher than that of the wild-type protein (LACS1).

By comparing the peak area, we found that the reaction rate of the mutant protein MLACS1 (BBa_K5255003) was nearly five times that of the wild-type LACS1 (BBa_K5255000) within two minutes of reaction (under 1mM DHA). This proves that the protein mutation is successful.

Based on the above content, we can preliminarily believe that we have obtained a protein with a higher affinity for DHA, and it can generate DHA-CoA in yeast. Our concept has been validated.

However, due to time and operational problems, we did not try and verify in Schizochytrium sp. A-2. We hope and encourage future iGEM teams to make further attempts and efforts on our project.

References

[1] K. Schneider, L. Kienow, E. Schmelzer, et al., Journal of Biological Chemistry, 2005, 280, 13962–13972.