Results
DNA construct creation
We successfully created 7 new basic (level 0) and 5 new composite (level 1) parts. (see Parts)
Basic parts
Basic parts were ordered from IDT and Twist Bioscience. These fragments were amplified with PCR using primers that are complementary to the Biobrick prefix and suffix.
- 1. Codon optimized zeaxanthin 7,8(7',8')-cleavage dioxygenase from Crocus sativus.
- 2. Codon optimized crocetin glucosyltransferase 2 from Crocus sativus.
- 3. Terminator from Rhodotorula toruloides (NP11) mRNA-heat shock 70kDa protein gene.
- 4. Codon optimized kanamycin resistance gene from Escherichia coli.
- 5. Promoter from Rhodotorula toruloides (NP11) glucose-6-phosphate isomerase (PGI) gene.
- 6. Codon optimized beta-carotene hydroxylase from Crocus sativus.
- 7. Codon optimized hygromycin resistance gene from Escherichia coli.
Composite parts
The composite parts were made using Golden Gate Assembly and then amplified using PCR with primers that add the necessary overhangs for level 2 assembbly
- TU1: PPGI + GLT2 + terminator
- TU2: PPGI + zcd + terminator
- TU3: PPGI + beta-carotene hydroxylase + terminator
- TU4: PPGI + kanamycin resistance + terminator
- TU5: PPGI + hygromycin resistance + terminator (not seen in the picture)
Medium optimization
UV-Vis measurements were used to compare total carotenoid content in R. toruloides extracts grown in different conditions. Absorption patterns were quite different, but some things could be noticed. First, growth at 20°C yields higher amounts of carotene than in 30°C in all cases. Second, extra lighting can boost carotenoid yields, but it can be noticed that change of light conditions does not affect all samples equally. Dramatical yield drop is detected in media 1 and 3, whereas changes in medium 2 between light and no-light (n) samples are almost negligible. Third, medium 4 gives lowest carotenoid yields in all conditions by a margin. As we were more interested in beta-carotene in our samples, we also ran an experiment for beta-carotene standard. The standard gave us two characteristic beta-carotene peaks at 452 and 483 nm. It can be seen that some samples present those two characteristic carotene peaks, especially samples from M2 medium, however the peaks are shifted bathochromically (460 and 490 nm, respectively). This shift can be easily explained: beta-carotene standard spectrum was recorded in dry hexane solution, whereas our extracts definitely had some water in them as well as residual acetone and DMSO from the extraction process, therefore making the solvent more polar and causing a bathochromic shift. Our very approximate estimates from UV-Vis spectroscopy suggest that at best growth conditions (M2, 20°C, extra light) R. toruloides produce beta-carotene at 1.5 mg/L concentration.
However, UV-Vis spectroscopy is more suitable for total carotenoid quantification than single carotenoid species detection. TLC analysis gave us more knowledge about types of carotenoids found in our extracts. It became clear that medium M2 was the best for beta-carotene production. Surprisingly to us, it turned out that M2 samples grown in 30°C actually had more beta-carotene than those grown in 20°C. Other mediums barely produced any beta-carotene, with M3 and M4 mediums at 20°C being second- and third-best carotene producers. From literature studies we know that the mildly polar compound giving the orange band in TLC plate is torularhodin. As can be identified, torularhodin is responsible for major part of absorption in M2 20°C samples. We could not identify the third major component on the extract with the highest polarity that did not move from the start line in hexane and in other TLC running solutions. This compound most likely has UV-Vis absorption peak at 520 nm as it is more characteristic to samples that contain less beta-carotene and torularhodin.
Beta-carotene elution time was determined to be at 4.57 min.
HPLC analysis was performed on five extracts.
The greatest yields of beta-carotene were achieved for R. toruloides grown in medium 2. It was also interesting to observe how the amounts of different carotenes - torularhodin and beta-carotene - varied among the different samples. For example, extracts from R. toruloides grown in M2 medium at 30°C had more beta-carotene than torularhodin, while the opposite was true for extracts from cells grown in the same M2 medium at 20°C. It seems like R. toruloides produces more beta-carotene at lower temperatures.
Uracil auxotrophic strain creation
R. toruloides seems to be semi-resistant to 5-FOA. The first generation of yeasts formed thick matts of colonies on the 5-FOA plates. After successional replica plating onto new 5-FOA plates, as well as SD plates with and without uracil, multiple possible URA3 mutants were isolated. Each colony was plated onto an SD plate with and without uracil to confirm the auxotrophy. None of the isolated mutants displayed inability to grow on SD without uracil, meaning they were not uracil auxotrophic.
Previous research suggests that not all 5-FOA resistant R. toruloides colonies in experiments like this are deletion mutants of URA3. Furthermore, the number of ‘false’ URA3 mutants can be high in proportion to all 5-FOA resistant colonies [1]. It has to be noted that this sort of random mutation induction causes random gene disruption. The albino phenotype of many mutants can be attributed to the deletion of CAR2 - gene encoding carotene cyclase without which beta-carotene is not formed in the cells [1]. For precise URA3 deletion CRISPR-Cas9 aided method can be used [2].
Transformations
We tried to transform our yeast with the DNA constructs we had created. Because R. toruloides prefers non-homologous recombination instead of homologous recombination, we tried exploiting this property and insert linear DNA fragments with no homology arms, hoping that it would insert in a place that doesn’t impact other physiological functions (see Notebook).
Before inserting other genes, it is important to know that our selection marker is working properly, in this case - our antibiotic resistance genes. We performed transformations with both TU4 (kanamycin resistance) and TU5 (hygromycin resistance). We performed the transformations, plate them on YPD plates for overnight growth and then the next day transfer the colonies over with a cloth on YPD plates containing the respective antibiotic.
For our TU4 transformations we observed no growth on plates containing kanamycin over 2 transformation attempts.
For our TU5 transformations in the second attempt we observed white colonies growing on plates containing hygromycin. Worried by the colonies not turning orange as the wild-type, we put the cells under a microscope. We observed that these were slow growing bacteria and that we have had contamination during our transformation process.
REFERENCES: [1] Koh, C.M.J., Liu, Y., Moehninsi et al. Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides. BMC Microbiol 14, 50 (2014). https://doi.org/10.1186/1471-2180-14-50 [2] Otoupal PB, Ito M, Arkin AP, Magnuson JK, Gladden JM, Skerker JM. Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides. mSphere. 2019 Mar 20;4(2):e00099-19. doi: 10.1128/mSphere.00099-19. PMID: 30894433; PMCID: PMC6429044.