Coupling Yeast and Mammalian Biosynthetic Pathways

.	The endogenous enzymes found in yeast have the ability to make Ergosterol. Mammalian enzymes genetically engineered into yeast can produce Hydrocortisone, using Ergosterol and Ergosta-5,7-dienol as substrates.
	Figure 5A: The Ergosterol synthesis pathway in yeast (yellow) was coupled to a genetically engineered hydrocortisone synthesis pathway (pink) using mammalian enzymes.

Reaction #	Enzyme	Native Species	Carbon #'s Affected	Reaction
1	Erg6p	Yeast	24,25	C-24 Methylation
2	Erg2p	Yeast	5,6,7,8,9	C8-9 Saturation, C5-6, C7-8 Desaturation
3	Erg5p	Yeast	22,23	C22-23 Desaturation
4	-7 Reductase	A.thaliana (plant)	7,8	C7-8 Desaturation
5	CYP11A1	Bovine	20	C20-22 Bond Cleaved, C20 Oxidized to Ketone
6	3-HSD	Bovine	3,4,5,6	C5-6 Saturation, C4-5 Desaturation, C3 Oxidized to Ketone
7	CYP17A1	Bovine	17	C17 -hydroxyl group added
8	CYP21A1	Human	21	C21 -hydroxyl group added
9	CYP11B1	Human	11	C11 -hydroxyl group added
Table 5A: Click on the reaction number (left) to link to the corresponding reaction.

Steriod Carbon Numbering

In each step of the biosynthetic pathway of hydrocortisone, one or more of the carbons of the sterol substrate are modified. The diagram on the right shows the structure of a cholesterol molecule with the carbons numbered.
	Figure 5B: A generic numbering scheme for sterols.

The first three steps in the biosynthesis of hydrocortisone are completed by endogenous yeast enzymes.

Zymosterol is the starting substrate in the Ergosterol syhthesis pathway in yeast. The full names of the enzymes Erg6p, Erg2p, and Erg5p are shown below: Abbreviation Enzyme Carbons Affected Erg6p S-adenosyl methionine -24 sterol-C-methyltransferase 24,25 Erg2p sterol C8-C7 isomerase 5,6,7,8,9 Erg5p 22(23)-sterol desaturase 22,23 Ergosterol and Ergosta-5,7-dienol are both used to as substrates for the next step in the biosynthetic pathway.

Figure 5C: The Ergosterol synthesis pathway in yeast (yellow) was coupled to a genetically engineered hydrocortisone synthesis pathway (pink) using mammalian enzymes.

 

Genetically-Engineered Hydrocortisone Synthesis Pathway

Reactions 4-9 in the biosynthesis of hydrocortisone are catalyzed by eight mammalian enzymes and one plant enzyme that were genetically engineered into yeast:

4)-7 Reductase

Figure 5D: 7-Rectuctase is the first non-native enzyme in this hydrocortisone biosynthetic pathway.

In this step, the double bond between carbons 7 and 8 is reduced. This reaction is catalysed by the 7-Reductase enzyme, which was genetically engineered into the yeast. 7-Reductase is a native enzyme of Arabidopsis thaliana, a small flowering plant The endogenous yeast enzyme Ncp1p (NADPH-P450 Reductase) supplies electrons to 7-Reductase One equivalent of NADPH is oxidised to NADP+ in this step

 

5) Cytochrome P450 side chain-cleaving (CYP11A1)

Figure 5E: CYP11A1 cleaves the bond between carbons 20 and 22, and oxidizes carbon 20 with a keto group.

In this step, the bond between carbons 20 and 22 is cleaved, and carbon 20 is oxidized to a ketone The CYP11A1 enzyme is a cytochrome P450 monooxygenase. The cDNA used was from bovine. Adrenodoxin (ADX) and Adrenodoxin Reductase (ADR) are necessary enzymes for electron supply to CYP11A1. ADR, ADX, and CYP11A1 are all normally located in the mitochondria, however, mature forms of these enzymes were expressed in the yeast, and CYP11A1 activity was present.

 

6) 3-Hydroxy steroid dehydrogenase/isomerase (3-HSD)

	The double bond between carbons 5 and 6 is reduced to a single bond, and a double bond is formed between carbons 4 and 5. The hydroxyl group on carbon 3 is oxidised to a keto group. This 3-HSD enzyme is of bovine origin.
	Figure 5F: 3-HSD saturates the C5-C6 bond and desaturates the C4-C5 bond. Also, the C3-OH is oxidised to a ketone group.
	Reaction 4 (17 hydroxylation) may occur before reaction 3 (3-HSD). In this situation, 17-OH pregnenolone is the substrate in the 3-HSD reaction, and 17-OH progesterone is the product. Note that the order of reactions 3 and 4 doesn't matter, as the net result is the same (the final product is 17-OH progesterone either no matter which reaction occurs first).
	Figure 5G: If 17-OH prenenolone is the substrate for 3-HSD, then 17-OH progesterone is the product.

 

 

7) Cytochrome P450 17-Hydroxylation (CYP17A1)

	This step is catalyzed by a Cytochrome P450 Hydroxylase A hydroxyl (-OH) group is added to carbon 17 with -stereochemistry (below the plane of the molecle). This CYP17A1 enzyme is of bovine origin. The endogenous yeast enzyme Ncp1p (NADPH-P450 Reductase) supplies electrons to CYP17A1.
	Figure 5H: CYP17A1, a Cytochrome P450 hydroxylase, adds an hydroxyl group to carbon 17.
	Reaction 4 (17 hydroxylation) may occur before reaction 3 (3-HSD). In this situation, pregnenolone is the substrate in the CYP17A1 reaction, and 17-OH pregnenolone is the product. Note that the order of reactions 3 and 4 doesn't matter, as the net result is the same (the final product is 17-OH progesterone either no matter which reaction occurs first).
	Figure 5I: If pregnenolone is the substrate for CYP17A1, then 17-OH pregnenolone is the product.

8) Cytochrome P450 21-Hydroxylation (CYP21A1)

	This step is catalyzed by a Cytochrome P450 Hydroxylase A hydroxyl (-OH) group is added to carbon 21 with -stereochemistry (below the plane of the molecle). This CYP21A1 enzyme is of human origin. The endogenous yeast enzyme Ncp1p (NADPH-P450 Reductase) supplies electrons to CYP21A1.
	Figure 5J: CYP21A1, a Cytochrome P450 hydroxylase, adds an hydroxyl group to carbon 21.

9) Cytochrome P450 11-Hydroxylation (CYP11B1)

This step is catalyzed by a Cytochrome P450 Hydroxylase of human origin. A hydroxyl (-OH) group is added to carbon 11 with -stereochemistry (above the plane of the molecle). The enzyme CYP11B1 is localized in the mitochondria. Instead of coupling this enzyme to its corresponding human reductase, scientists instead coupled it to a natual yeast reductase Arh1p and the bovine Adrenodoxin (ADX) electron carrier.

Figure 5K: CYP21A1, a Cytochrome P450 hydroxylase, adds an hydroxyl group to carbon 21.