Our goal was to engineer the progesterone pathway genes into a plasmid and produce progesterone in Y. lipolytica. We show evidence of progesterone production in our engineered Y. lipolytica strain under real-world growth conditions.
We used yeast-mediated cloning (YMC) to assemble the plasmid pOPPY-XRL2-yP. To select for successful plasmid assembly in our yeast Y. lipolytica str. FKP393 (matA; leu2-270; ura3; xpr2-332; axp-2; ku70::hph+), we used the auxotrophic marker leucine. We inserted the individual progesterone pathway genes ∆7-sterol reductase, ADR, FDX1, P450scc, 3β-HSD, and using YMC, we assembled them with the linearized plasmid backbone pXRL2 (leu+) to produce the circularized plasmid product in Y. lipolytica.
Figure 1: We will linearize the pXRL2 plasmid using inverse PCR to remove one loxP site (loxP cutout). We will re-circularize and re-linearize the plasmid with one loxP site, pOPPY-XRL2-yX. We will insert the genes ∆7-sterol reductase, ADR, FDX1, P450scc, and 3β-HSD, each of which has a TEF1 promoter, a Tsynth8 terminator, and Gibson overhangs (each denoted by GO) to assemble the fragments together, and into the plasmid backbone during YMC to create pOPPY-XRL2-yP.
These cells produced colonies on leucine-deficient media, demonstrating that our plasmid pOPPY-XRL2-yP was successfully assembled. We miniprepped these plasmids and ran them on a gel; the results showed that the plasmids were above 15kb, which is the correct size for the plasmid containing the pathway genes with the plasmid backbone. This further suggests that we successfully assembled our plasmid.
Figure 2: We performed yeast mediated cloning to assemble delta 7-sterol reductase, ADR, FDX1, P450scc and 3ꞵ-HSD into pOPPY-XRL2-yX. We plated the transformants and saw many colonies. We grew 30 colonies selected from leucine-deficient plates in leucine-deficient liquid media to mini-prep them and check for successful plasmid assembly. We rescued the plasmid and ran 250ng dilutions of our samples on a gel. The expected size of the desired plasmid with all pathway genes, pOPPY-XRL2-yP, was approximately 15kb in comparison to pXRL2 which is 6693bp. All of the samples ran were circularized plasmids which is evident in the bands occurring higher than the ladder since they run slower. Samples 1, 2, 9 and 14 had bands higher than the plasmid above 10kb so we selected them for sequencing to confirm their success.
To test these colonies for progesterone, we grew six colonies in leucine-deficient liquid media, then performed the Progesterone Extraction protocol listed on our protocols page. We “dot blotted” the progesterone, purified it out of our cells on nitrocellulose membranes, and detected it using anti-progesterone antibodies conjugated to HRP (sc-53423 HRP) which was gifted from Santa Cruz Biotech (Santa Cruz Biotechnology, Inc., TX, USA). Our results were promising.
Figure 3: Dot blot of progesterone re-suspended in DMSO, isolated from colonies grown on leucine-deficient media after YMC with progesterone pathway genes. Negative control circled in black and marked with a dash (-). Positive controls circled in blue. Experimental samples circled in pink. Primary antibody used was anti-progesterone-HRP, blocked with BSA in TBS, washed with TBS/TWEEN. Positive controls are 1mg/100uL progesterone in DMSO with 1:100 dilutions. Negative control is Y. lipolytica transformed with pD17 with no pathway genes which underwent same extraction protocol as all other samples.
Our dot blot shows that we have high luminescence in multiple colonies, YL 10, YL 20, YL 21, and YL 25. We performed the extraction protocol on +1 positive control, and our negative control. The +1 positive originally contained “1mg” of pure progesterone extract from Sigma Aldrich (P8783 Sigma), 1mL of MilliQ water, and 1mL of hexane which we performed the Progesterone Extraction protocol on.The positive controls +3, +4 and +5 are 1:10 dilutions from +2 positive control of 1mg/1mL of pure progesterone in DMSO. These positives were not subjected to the extraction protocol. We believe that they may show no luminescence due to +2 being one order of magnitude less than detection is possible for the antibodies. The negative control is pD17 transformed into Y. lipolytica with no pathway genes, and then subjected to the same extraction protocol.
These results suggest that we have at least four progesterone-producing Y. lipolytica colonies. The positive control shows the level of luminescence for 1mg of progesterone, and the colonies that show luminescence show a similar magnitude.
While we were not able to insert the progesterone biosynthesis pathway into the chromosome of our Y. lipolytica, we believe that we have shown that the assembly of a five-gene pathway via YMC to produce progesterone is possible, and therefore works under real world conditions.
Due to the ability of our hexane extraction protocol to considerably purify our samples, we will perform mass spectrometry on these samples in later work and continue to work on engineering the pathway directly into the genome.