Experiments: The Progesterone Producing Yeast Strain

Experiments: the progesterone producing yeast strain Our team will use synthetic biology to address insufficient access to contraception by engineering a progesterone-producing yeast. We will construct our gene cassettes and insert them into the Yarrowia lipolytica genome in three parallel experiments. In the preliminary experiment, we will use the strain Y. lipolytica str. FKP393 which has the auxotrophic markers leu2 and ura3 for selection. We will engineer a second strain named LipLox, that will contain the gene for uracil flanked by lox sites loxP and lox71 for use in experiments 1, 2, and 3 below. This will allow us to have a yeast strain that contains lox sites, as well as a strain that now has uracil in a designed location that we can swap in or out as a selectable marker.

The first step in our project was to engineer a strain of yeast with an genetic marker in the location we plan on inserting the genes for the progesterone biosynthesis pathway. We also use Cre-lox integration in later experiments, so we needed to enter lox sites into our strain as well. Because Y. lipolytica str. FKP393 is auxotrophic for uracil, we can replace this gene in a specific location as a marker for a second homologous recombination (HR) experiment. When we perform the other HR experiment, the uracil gene will be swapped out, and we can use 5-FOA media as our selection tool.

Homology Arms

We designed homology arms (HA) for homologous recombination that span 1 kb upstream and downstream of the ade2 gene in Y. lipolytica str. FKP393 . To prepare HAs for this experiment, and experiment 1, we amplified the HAs out of the Y. lipolytica str. FKP393 genome using primers flagged with Gibson overhangs (GOs) homologous with the gene cassette ends and the linearized pUC19 backbone as seen in Figure 1. This ensured that the HAs were ready for GA after being amplified out.

Figure 1: Isolation of DNA from yeast, and PCR amplification of upstream and downstream homology arms that surround ade2 out of Y. lipolytica str. FKP393. Primers with Gibson overhang flags are used to create homology arms with Gibson overhangs on each end that associate to their corresponding fragments.

We performed colony PCR to amplify the HAs. After several failed attempts, we found that we needed to extract the genomic DNA (gDNA) from Y. lipolytica str. FKP393 and then PCR-amplify the HAs out of it. To extract the gDNA from Y. lipolytica str. FKP393, we used lithium acetate and sodium dodecyl sulfate (SDS) to lyse the cells, then precipitated the gDNA with ethanol. See our Protocols section below for Yeast DNA Extraction protocol. We used touchdown PCR with the gDNA and flagged primers to amplify the arms out. We checked the success of PCR amplification using gel electrophoresis.

Enter lox sites and ura3 into Y. lipolytica str. FKP393 genome

To create LipLox, which contains the two lox sites used in experiments 2 and 3, as well as the ura3 gene that will act as a selective marker in all experiments, we will Gibson assemble the pOPPY-UC19-yXXU plasmid shown in Figure 2.

Figure 2: pOPPY-UC19-yXXU plasmid in detail. Contains UHA, terminator Tsynth8, loxP site, TEF1 promoter, ura3 gene, Tsynth8 terminator again, lox71 site, and DHA. Will be used to insert lox sites and ura3 into Y. lipolytica str. FKP393.

This plasmid contains our loxP-ura3-lox71 gene block, surrounded by the UHA and DHA to Y. lipolytica str. FKP393 genome, in the linearized pUC19 backbone. The HAs were designed with ends homologous with loxP-ura3-lox71 gene block and pUC19. This design enables GA to seamlessly stitch together all four fragments resulting in pOPPY-UC19-yXXU seen in Figure 2. Because HR requires a linear construct, we will amplify out the product from UHA to DHA. We will perform HR and the loxP-ura3-lox71 fragment will be exchanged for the ade2 gene region between the HAs in the Y. lipolytica str. FKP393 genome (Figure 3).

Figure 3: Creation of pOPPY-UC19-yXXU containing ura3, pUC19 backbone, lox sites and homologous arms using Gibson Assembly. Transformation of Y. lipolytica to create LipLox using homologous recombination.

The yeast recombinants, which were previously auxotrophic for uracil, will now contain ura3 and will grow on uracil-deficient media, allowing us to select for them. As a second method of confirmation, we will extract DNA from transformed Y. lipolytica str. FKP393 cells and use PCR to screen for successful mutants. We will know that we have successful HR results if we observe single bands at the appropriate size (4 kb) on a 1% agarose gel.

Experiment 1 will use well-studied methods previously tested in Y. lipolytica str. FKP393. We will assemble the plasmid pOPPY-UC19-yP to create the construct necessary to integrate into the LipLox genome to produce Y. lipolytica str. PoPPY. We will Gibson-Assemble the linearized pUC19 plasmid backbone, the UHA and DHA amplified out of the Y. lipolytica str. FKP393 genome, and the five progesterone pathway genes to create pOPPY-UC19-yP seen in Figure 4.

Figure 4: pOPPY-UC19-yP plasmid in detail. Contains UHA, DHA, and the genes ∆7-sterol reductase, ADR, FDX1, P450scc, and 3β-HSD, each of which has a TEF1 promoter, Tsynth8 terminator, and Gibson overhangs (each denoted by GO) to assemble the fragments together, and into the plasmid backbone. Will be used to engineer LipLox with the progesterone pathway. BioBrick prefix and suffix are described in “BioBrick” section.

Figure 5: Creation of pOPPY-19-yP via GA of progesterone biosynthesis pathway genes, HAs, and linearized pUC19 backbone. Transformation of LipLox using HR to exchange gene cassette. Selection of PoPPY using 5-FOA ura+ media.

HR requires a linear fragment, so we will amplify out our construct from UHA to DHA and perform HR to recombine the construct into the LipLox genome. HR will exchange the previously inserted ura3 from LipLox with the gene cassette from pOPPY-UC19-yP. The new yeast strain, named Y. lipolytica str. PoPPY, will be uracil-deficient again, so we will test for successful integration by growing the yeast on 5-Fluoroorotic acid (5-FOA) ura+ media to select for our new strain containing our gene cassette as seen in Figure 5.

Experiment 2 will combine two well-studied experimental techniques. Yeast mediated cloning, which can assemble fragments with Gibson overhangs inside of a yeast cell; and Cre-lox recombination, which can perform a homologous-recombination-like insertion where there are corresponding lox sites. We will use YMC in S. cerevisiae, due to that technique being well studied in that organism, to create the plasmid pOPPY-XRL2-yP (Figure 6) that contains the gene cassette required to complete the progesterone biosynthesis pathway. We will isolate this plasmid and then transform it into LipLox.

Figure 6: 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.

We will linearize pXRL2 using PCR and remove one of the two loxP sites to produce pOPPY-XRL2-yX. We will re-circularize pOPPY-XRL2-yX before re- linearizing with PCR primers in a region that is homologous with both ends of the gene cassette. We will insert a re-linearized pOPPY-XRL2-yX with the five pathway genes into S. cerevisiae for yeast mediated cloning. We will miniprep out the pOPPY-XRL2-yP plasmid and transform into E. coli. We will insert our gene cassette into the LipLox genome using Cre-lox with lox sites entered in experiment 0 to produce Y. lipolytica str. PoPPY as shown in Figure 7.

Figure 7: Creation of pOPPY-XRL2-yP via YMC in S. cerevisiae using linearized pXRL2 and gene fragments. Transformation of Y. lipolytica str. LipLox using pOPPY-XRL2-yP followed by Cre-lox recombination and 5-FOA ura+ selection to create Y. lipolytica str. PoPPY.

We will amplify pOPPY-XRL2-yP in E. coli and isolate the plasmid using the ZyppyTM Plasmid Miniprep Kit from Zymo Research. We will transform pOPPY-XRL2-yP and the pSH47 plasmid that contains the genes for Cre recombinase into LipLox and perform Cre-lox recombination. The pXRL2 plasmid contains one loxP site upstream of the gene construct, and the downstream end of the gene construct contains the lox71 sequence necessary for Cre-lox recombination. The presence of galactose will induce the expression of Cre recombinase and trigger site-specific recombination between the incompatible lox71 and loxP sites on pOPPY-XRL2-yP and in the LipLox genome to make PoPPY as in Figure 7. The five pathway genes on pOPPY-XRL2-yP and ura3 gene in LipLoxura3 gene through non-selective screening and allow us to select for our desired cells using 5-FOA.

Experiment 3 will be a novel undertaking; while YMC has been tested in Yali, Cre-lox has not. In Experiment 3, we will use YMC in LipLox to create the plasmid pOPPY-XRL2-yP (Figure 6) to insert our gene cassette into the LipLox genome to produce Y. lipolytica str. PoPPY, shown in Figure 8.

Figure 8: LipLox will create pOPPY-XRL2-yP from the five pathway genes and pOPPY-XRL2-yX in YMC. Cre recombinase will induce recombination between the lox sites on the plasmid and the lox sites in the genome to create PoPPY.

We will insert pOPPY-XRL2-yX with the 5 pathway genes into LipLox to create pOPPY-XRL2-yP (Figure 6). The pXRL2 plasmid contains one loxP site upstream of the gene construct, and the downstream end of the gene construct contains the lox71 sequence necessary for Cre-lox recombination. We will amplify pOPPY-XRL2-yP in LipLox, wait for YMC to completely assemble the construct, then transform the Cre recombinase gene-containing pSH47 plasmid into LipLox to perform Cre-lox recombination and create PoPPY.

Protocols