Two different Module 1 plasmids were created.
Coding sequences for DHCR7 and were ordered in an IDT Golden Gate vector. In a first round of GG-Assemblies, these were combined by BbsI with promotors and terminators and inserted into a Backbone with AMP resistance cassette and cloning sites which allows to combine the genes into one cassette.
| Gene | Promotor | Terminator | Backbone |
|---|---|---|---|
| DHCR7 | pGAP | RPL2Att | BB2_AMP_AB |
| DHCR24 | pTEF(569) | RPP1Btt | BB2_AMP_BC |
After transformation int E. coli, Mini prep and restriction digest and gel electrophoresis to confirm correct assembly, these expression cassettes were assembled via BsaI into a Backbone with NTC resistance which allowed integration into the pRGI site of the Genome of Pichia pastoris (Plasmid was called p16) Correct assembly wasascertained by transformation, mini-prep, restriction digest and gel electrophoresis.
For a different strain an alternative Module 1 was created. For this, DHCR7 was combined with Bbs1 with pGAP and RPL2Att and directly inserted int a Backbone with NTC resistance and which allowed integration into the pRGI site of the Genome of Pichia pastoris (Plasmid was called p2) Correct assembly was ascertained by transformation, mini-prep, restriction digest and gel electrophoresis.
Coding sequences for CYP11A, Adrenodoxin and Adrenodoxin reductase were Ordered in an IDT Golden Gate vector. In a first round of GG-Assemblies, these were combined by BbsI with promotors and terminators and inserted into a Backbone with AMP resistance cassette and cloning sites which allows to combine the genes into one cassette.
| Gene | Promotor | Terminator | Backbone |
|---|---|---|---|
| CYP11A | pGAP | RPL2Att | BB2_AMP_AB |
| Adrenodoxin | pTEF(569) | RPP1Btt | BB2_AMP_BC |
| Adrenodoxin reductase | pMDH3 | RPS25Att | BB2_AMP_CD |
After transformation int E. coli, Mini prep and restriction digest and gel electrophoresis to confirm correct assembly, these expression cassettes were assembled via BsaI into a Backbone with HYG resistance which allowed integration into the ENO site of the Genome of Pichia pastoris. (Plasmid was called p20) Correct assembly was again ascertained by transformation, mini-prep, restriction digest and gel electrophoresis.
Coding sequences for CYP17A1, HSD3B1 and HSD17B3 were ordered. In a first round of GG-Assemblies, these were combined by BsaI into a Backbone with KAN resistance and cloning sites wich allow attachment of promotor and terminator fragments. Correct assembly was ascertained by transformation, mini-prep, restriction digest and gel electrophoresis.
In a second step the coding sequences were combined by BbsI with promotors and terminators and inserted into a Backbone with AMP resistance cassette and cloning sites which allows to combine the genes into one cassette.
| Gene | Promotor | Terminator | Backbone |
|---|---|---|---|
| CYP17A1 | pGAP | RPL2Att | BB2_AMP_AB |
| HSD3B1 | pTEF(569) | RPP1Btt | BB2_AMP_BC |
| HSD17B3 | pMDH3 | RPS25Att | BB2_AMP_CD |
After transformation int E. coli, Mini prep and restriction digest and gel electrophoresis to confirm correct assembly, these expression cassettes were assembled via BsaI into a Backbone with ZEO resistance which allowed integration into the AOXTT site of the Genome of Pichia pastoris. (Plasmid was called p21) Correct assembly was again ascertained by transformation, mini-prep, restriction digest and gel electrophoresis.
After transformation int E. coli, Mini prep and restriction digest and gel electrophoresis to confirm correct assembly, these expression cassettes were assembled via BsaI into a Backbone with HYG resistance which allowed integration into the ENO site of the Genome of Pichia pastoris. (Plasmid was called p20) Correct assembly was again ascertained by transformation, mini-prep, restriction digest and gel electrophoresis.
Pichia pastoris CBS 7435 were transformed by electroporation (r 2 kV, 25 µF and 186 Ω) with 7,5 mg each of p2, p20 and p21. The plasmids p2 and p21 were linearized by digestion with AscI, p20 with SmaI. Selection after transformation was done on YPD Agar with Nourseothricin (100 µg/mL), Hygromycin (100 µg/mL) and Zeocin (25 µg/mL), by incubation at 28°C for 72h.
Pichia pastoris CBS 7435 ΔErg6 were transformed by electroporation (r 2 kV, 25 µF and 186 Ω) with 10 mg each of p16, p20 and p21. The plasmids p2 and p21 were linearized by digestion with AscI, p20 with SmaI. Selection after transformation was done on YPD Agar with Nourseothricin (100 µg/mL), Hygromycin (100 µg/mL) and Zeocin (25 µg/mL), by incubation at 28°C for 96h. Only two colonies appeared.
Cells for the determination of the Testosterone producing capabilities were grown in shake flask cultures. 10 mL of YPD with Nourseothricin (100 µg/mL), Hygromycin (100 µg/mL) and Zeocin (25 µg/mL) for the modified strains or without Antibiotics for the unmodified strains were prepared in shake flasks and inoculated with 0,1 OD units from precultures. After one to two days, when sufficient biomass had accumulated, the cultures were harvested to portions of 15 OD units.
The extraction for Sterol determination was done according to a method published by Ott et all 2005 [1]. Cells amounting to 15 OD units were suspended in 0,4mL methanol, 0,6 mL 0,5% pyrogallol in methanol and 0,4 mL 60% aqueous KOH. After thorough mixing the centrifuge tubes were placed on a 90°C shaking heating block for 2 h. The residue was thrice extracted with 1 mL of heptane, which was evaporated at 105°C in a heating block.
To determine whether this extraction method was able to extract relevant quantities of sterols from our sample recovery rate experiments were conducted. For this, samples were spiked with 20 µg of Ergosterol, Cholesterol, Pregnenolone acetate and Testosterone, once before the digestion, once after the extraction.
The extracts were derivatized in a reaction with 40µL Pyridine, 40µL BSTFA with 1% TMCS and 120 µL ethyl acetate. The reaction was heated to 85°C for 60 min.
For Analysis a Macherey-Nagel OPTIMA5-MS accent 60 m x 0.25 mm x 0.25 µm with an oven program of 100°C (0.5 min), 35°C/min to 255°C, 3°C/min to 305°C (7 min). Carrier gas was Helium with a flowrate of 1,2 mL/min. 1µL of sample was injected in a 290°C splitless inlet with a single taped liner. The mas spectrometer measured both in SIM and Scan. Scan was conducted from 50-500, while SIM used the masses 270, 298, 360, 363, 368, 388, 458, 468. The ion source was heated to 230°C.
An Agilent 1200 HPLC system was used, comprising of binary pump, degasser, autosampler and column oven. The detector is an evaporative light scattering detector ELSD Sedex 85 of Fa. Sederle. The Column used was a Luna C18 4,6x150 mm, 5µm from Phenomenex. As eluents 0,05% aqueous TFA (A) and 0,05% TFA in isopropanol (B). The following gradient was used at a flow rate of 0,65 mL/min.
| Time [min] | %B |
|---|---|
| 0 | 75 |
| 6,25 | 80 |
| 10 | 90 |
| 11 | 100 |
| 15 | 100 |
| 16 | 75 |
| 21 | 75 |
The colum was heated to 50°C, the detector to 40°C, with a gain factor of 12. The sample after complete evaporation was dissolved in 200 µL Methanol of which 25 µL were injected.
A rough measure of the growth of the produced strains was desired to contrast it to the strain Testosterone production capabilities. Cultures of unmodified wildtype, unmodified Erg6-KO, modified wildtype and modified Erg6-KO were prepared. 10 mL YPD media without antibiotics was aliquoted into 100 mL shake flasks and inoculated with cell suspension to an initial OD of 0,05. The inoculum was taken from liquid pre cultures grown over the weekend. The were incubated at 25°C on a shaker at 180RPM. Growth was measured as OD600 after 6,5h, 10,5h, 23,5h and 33h.
It was planned to produce Erg5-KO strains, for which a KO cassette needed to be constructed. First, the flanking regions of the Gene were amplified with overhang Primers, then the KAN-MX-cassette with complementary primers. A fusion of these fragments however was unsuccessful.