YEAST MEDIATED CLONING

Due to our extensive research on world issues, we chose to create a contraceptive in yeast: progesterone. Progesterone is a hormone produced by all mammals (men and women) naturally, and aids in many functions regulating fertility, menstruation/ovulation, pregnancy, and normal body function. "Progesterone prepares the endometrium for the potential of pregnancy after ovulation. It triggers the lining to thicken to accept a fertilized egg. It also prohibits the muscle contractions in the uterus that would cause the body to reject an egg. While the body is producing high levels of progesterone, the body will not ovulate" (hormone.org). Progestins are routinely used in contraceptive pills, vaginal rings, and IUDs. Progestins are a chemically formulated version of progesterone (CITATION!!!), that acts on progesterone receptors to increase cervical mucus and suppress ovulation to prevent pregnancy. They are not naturally made by the human body, and therefore have many ill effect on the women who take them (CITATION!!!!!). Progesterone is the molecule naturally made by the body, which when taken in excess, can perform the same function as progestins: preventing pregnancy. Progesterone can do so without the harmful effects that progestins cause (CITATION!!!!). In mammals, the progesterone biosynthesis pathway begins with cholesterol. Enzymes convert cholesterol to pregnenolone to progesterone. Figure 1 shows a highly simplified version of the reference pathway for steroid hormone biosynthesis in mammals from the Kyoto Encyclopedia of Genes and Genomes (KEGG) to highlight the progesterone biosynthesis pathway.

The organism we are engineering, Y. lipolytica, does not produce cholesterol naturally, but it does produce a cholesterol precursor: zymosterol. In Y. lipolytica however, only the zymosterol-ergosterol pathway exists. We considered engineering three non-native enzymes (circled in blue in Figure 2) into Y. lipolytica to complete the zymosterol-cholesterol pathway, due to the common pathway to progesterone in mammals starting from cholesterol. However, because ergosterol is the yeast’s preferred pathway product, navigating through the cholesterol pathway would require completely knocking out the pathway to ergosterol (starting from ERG6 on the right). The addition of those three genes, plus the additional genes needed to take cholesterol to progesterone, PLUS a pathway knockout required too much engineering for the timeframe of our project. The enzymes highlighted in green exist in Y. lipolytica, see the end product of ergosterol circled in pink. The enzymes highlighted with white do not exist, and would need to be engineered into the organism.

We needed to figure out how to create progesterone in a creative way. We found that we could create a synthetic pathway to progesterone, directly from ergosterol (circled in pink in Figure 2). This was beneficial because that pathway exists naturally in Y. lipolytica. In Y. lipolytica and other fungi, ergosterol serves similar functions in fungi as cholesterol does in animal cells (Money, 2016), meaning that while the pathway does not currently exist in the organism, it is possible to convert ergosterol to progesterone. We searched through research articles and found the work of Duport et al.(CITATION??!!) who outlined the possible pathway to convert ergosterol to progesterone using five genes. The first gene is for the enzyme ∆7-sterol reductase (∆7Red), isolated from Arabidopsis thaliana, which reduces ergosterol to ergosta-5-enol and ergosta-5,22-dienol (Waterham and Wan- ders, 2000). The next gene encodes for bovine side-chain cleavage cytochrome (P450scc), which converts ergosta-5-enol and ergosta-5,22-dienol to pregnenolone. P450scc requires the assistance of bovine ferredoxin-1 (FDX1) as an electron carrier (Grinberg et al., 2000), and adrenodoxin reductase (ADR) to reduce FDX1. The final gene is type II human 3β-hydroxysteroid dehydrogenase (3β-HSD) that converts pregnenolone to progesterone (Koritz, 1964). See the complete pathway to progesterone biosynthesis in Y. lipolytica below in Figure 3.

We found the sequences for each gene from either KEGG or UniProt and codon optimized them for Y. lipolytica using the DNA works system from the High-Performance Computing at the NIH website.