iGEM 2023 — Team boku-vienna

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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##### Content

* [Weekly Notebook](#Weekly Notebook)
* [Scanned Notebook](#Scanned Notebook)

## Weekly Notebook

## Scanned Notebook

### Notebok Part 1

### Notebok Part 2

### Notebok Part 3

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{% block title %}Engineering Success{% endblock %}
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##### Content

* [Introduction](#Introduction)

## An illustration of the DBTL Cycle:

The Engineering Design below, represents the ideal that our team strives toward, the first step of the DBTL Cycle. However, the point of the cycle is that things become modified in contact with reality. (To see how the Engineering turned out this cycle check out the Results)

During the project many successful project milestones were completed and built, but may have struggled when put to the test, others behaved much better than could have been expected! We’ve built, tested, and learned our way through this project, but let’s look at a specific example and see what we’ve learned.

In theory It is possible to design a process that is perfectly planned, but not with such a complex project and such a small lab team. With only 3 students approved by the university to work in the lab, and only 2 available for much of the summer we had to get creative with our the implementation of or engineering and design.

Finishing the long road to completed golden gate cloning of our multi-plasmid system by the end of August. Knowing our limitations we originally tried to avoid cloning entirely by working with a corporate sponsor for complete plasmid synthesis, but they couldn’t accommodate us. Speaking to our PI’s the lab team calculated 2-5 days for transformation, then 2-5 days for competency, per plasmid. With 4 plasmids total per strain, we would have to remove a plasmid with Lox, all while fighting drawdown effects from the Pichia. In total an additional 2-3 weeks, if everything was successful. A potentially feasible amount of time, our professors advised. Sometimes the willingness to take a shot in the dark pays off however. While performing the sequential experiments as planned, our wet lab captain Simon decided to see what would happen if you just transformed a strain with all 3 plasmids at once. We knew that uptake of DNA was incredibly rare, so it happening 3 times to the same yeast seemed almost impossible, but it only had to happen once. 1 week later we had seeming success! A colony growing on our multi-antibiotic plate! But how to verify transformation?

According to our literature research at the time, the knockout of the gene that catalyzes the final step of the ergosterol pathway (ERG5) was a prerequisite for steroid production. To that end, we designed a process for knocking out this gene through colony PCR. The plan was to design PCR primers to extract flanking regions and then fuse them with a selection marker cassette. In the end we were not successful in performing the ERG5 knockout in the time we had because of issues with the fusion cassete. That doesn’t mean we didn’t learn from the experience. After a seemingly successful multi-plasmid transformation step we needed a way to verify transformation. Remembering our experiences with primer design, we knew colony PCR could be the answer! Designing primers was easy with our previous experience, but again we were frustrated by circumstance. Previous primers had arrived within a week, but while performing our final order with IDT we added extra enzymes which have delayed the process enough that as of wiki freeze they have not arrived. In the end I guess we can learn from that too!

## Introduction

Souza and colleagues1 have replaced ergosterol, which has essential function in modulating membrane physical properties, membrane protein stabilisation and localisation, as well as physiological functions such as endocytosis, protein sorting, and support receptor protein activities in P. pastoris, with cholesterol, a biological and biochemical functional counterpart of ergosterol in animalia 1,2. In doing so, they deleted Erg6 (sterol 24-C-methyltransferase), Erg5 (sterol 22-desaturase) genes, and inserted DHCR7 (Danio rerio 7-dehydrocholesterol reductase) and DHCR24 (Danio rerio delta24-sterol reductase) genes. Importantly, they have shown that their erg5Δerg6ΔDHCR7+DHCR24+ strains are stable and able to produce cholesterol, with a yield of about 1 mg cholesterol per gram wet weight of P. pastoris, using alkaline methanolysis, petroleum ether extraction, and C18 HPLC purification. Could we go further and make it through the steroid pathway up to testosterone? Another important work was the iGEM team 2018 UCSC, who synthesized progesterone de novo aiming at making birth control more accessible. An interesting feature of their work was a riboswitch that would limit the end concentration of the hormone in the fermentation broth and thus mitigate the risk of overdosage upon misuse. An important foundation for their progesterone synthesis pathway was the research done by Duport et al. 1998, who described de novo progesterone synthesis in S.cerevisiae. Since progesterone is a precursor of testosterone, we could build on this already explored synthesis pathway, leaving only the further path from progesterone to testosterone uncharted waters, or at least we thought so initially. Later we learned that Szczebara et al. (2003) have reported the first total de novo synthesis of the steroid hydrocortisone from simple carbon sources in S.cerevisiae, building on the previously by Duport et al.(1998) reported de novo progesterone synthesis process. Their hydrocortisone process would star two of the three enzymes required for testosterone synthesis, leaving only the branch to testosterone open – an enzymatic conversion that we later learned that has already been reported in a stand-alone application as well (Shao et al., 2016). Our work would therefore recombine the work of those publications into a novel path. What further distinguishes our approach from previous work is as follows: While the iGEM group 2018 UCSC aimed at low concentrations of progesterone, we, the iGEM BOKU team 2023, aim at optimizing steroid concentration to make a competitive industrial process for testosterone. Therefore, we not only inserted the enzymes required to produce the hormone, but also aimed to explore the strain development of Duport et al. 1998 that involved the deletion of a native yeast enzyme, thus not having the yeast’s native ergosterol but also its Δ22 saturated form as starting point for sterol synthesis. As a third strain development strategy we planned to use the cholesterol process reported by Souza et al. in 2011 and by Hirz et al. 2013 as basis for the further steroid pathway – a strain development option that has not been reported so far to the best of our knowledge.

## IDENTIFICATION OF GENES AND PLASMID DESIGN

### Host organism

As host organism, we chose the yeast Komagataella phaffii (formerly Pichia pastoris), strain number CBS7435, kindly provided by BOKU. Up to date, a commonly used organism for production of steroid hormones has been Mycobacterium sp . Mycobacteria can take up sterols such as cholesterol from the environment and grow on them as carbon source, as well as have been shown to natively convert cholesterol and other sterols into steroids. Fernández-Cabezón et al. have used Mycobacterium smegmatis for testosterone production starting from phytosterols, from cholesterol, and from androst‐4‐ene‐3,17‐dione (AD). With M. smegmatis they had chosen a Mycobacterium strain without native ability to metabolize or degrade the AD and testosterone, so that the products would be stable, and inserted the gene for HSD17B, which converts AD to testosterone, recombinantly . The strain of our choice, K.phaffii likewise does not possess the set of enzymes to metabolize AD and testosterone according to the KEGG pathway , except for a hypothetical protein that might convert AD to androstanedione. While mycobacteria are used for their ability to take up cholesterol from the environment, we aimed to avoid the need for cholesterol feeding by exploiting the yeast’s endogenous sterol production. This allows for using simple, cheap and abundantly available carbon sources such as glucose and beyond. This would not be possible in bacteria such as Mycobacterium sp. due to the lack of endogenous sterols in bacterial membranes in general (Huang and London 2017) . To decide which yeast strain we would take, first, we considered S.cerevisiae because it was used by Souza et al (2011) , who succeeded in giving the organism a cholesterol containing membrane. Another consideration was Y. lipolytica, inspired by the iGEM team 2018 of UCSC . Eventually we chose K.phaffii to be our workhorse, since the workgroup of our iGEM supervisors has a strong track of using K.phaffii and would be able to provide us with a variety of resources such as K.phaffii strain CBS7435, Golden Gate cloning plasmids with genome integration sides for K.phaffii, respective primers, and more. Another factor endorsing the choice of K.phaffii was that upon further literature research, we came across the paper of Hirz et al. (2013) who showed that the membrane of K.phaffii can be modified towards replacement of ergosterol by cholesterol in the yeast’s membrane as well, similar to the work of Souza et al. 2011. This was information for us because it shows that our strains would be able to survive with modified sterol content in the membrane. Further, Shao et al. 2016 have described the transformation step of AD to testosterone by insertion of a HSD17B gene into K.phaffii (formerly P.pastoris), proving that the yeast would grow in presence of testosterone. Therefore, K.phaffii would be a good option for the scope of our project.

### Experimental design

Our process comprises nine heterologously expressed enzymes organized on three inserted plasmids, further referred to as Module 1, Module 2, and Module 3, as well as two gene deletions (Fig.1a). A detailed overview about the strain development can be seen in Fig.1b,c, while further elaborations of the choice of genes and gene deletions is stated below.

![](https://static.igem.wiki/teams/4700/wiki/images-enginering/9ec0ef2e-008e-4eb9-9418-796db0b6a8a6.jpg)

### Module 1 genes

In our process, in addition to an established stable cholesterol producing platform (erg5Δerg6ΔDHCR7+DHCR24+) by Souza et al.1, other groups (see Fig.1b, c, f and below), were evaluated. The establishment comprises 3 alternatives in total: 1a), erg5Δerg6ΔDHCR7+DHCR24+, as described and characterised in Souza’s 2011 paper1; 1b), erg5Δerg6+DHCR7+, as described and characterised in Duport et al.3 in Saccharomyces cerevisiae; and 1c), erg5+erg6+DHCR7+ as attempted by the University of California Santa Cruz 2018 iGEM team (see Table.1) in Yarrowia lipolytica. To make those strains, two different versions of the Module 1 genes were constructed: Module 1a comprising DHCR7 and Module 1ab comprising both DCHR7 and DHCR24. cDNA was taken from danio rerio because this has been shown by Souza et al 2011 to work best out of several options in S.cerevisiae, and was subsequently also shown to work in K. phaffii by Hirz et al 2013 .

Zymosterol is the shared precursor of ergosterol (Erg6, Erg2, Erg3, Erg5, Erg4, referred to as Erg2-6 pathway below) and cholesterol (EBP, SC5DL, DHCR7, and DHCR24 pathway Fig.1f)2, consistently, Gu et al.4 advised that ergosterol synthesis pathway competes with exogenous cholesterol biosynthetic pathway. Importantly, erg5Δ and erg6Δ strains are viable4,5,6,7. Thus, our strain A would have enhanced cholesterol biosynthesis given that Erg2 (Δ8, Δ7 isomerisation) and Erg3 (C5 desaturation) share the same function with EBP and SC5DL, respectively. The sole deletion of ERG5 in Erg2-6 pathway was argued to enhance pregnenolone production by minimising desaturation at the carbon 22, making a characteristic Ergosta-5-ene-ol, which is thought to be a better substrate for the module 2 p450scc (CYP11A1)3,8. Thus, in our strain B, Ergosta-5-ene-ol instead of cholesterol serves as a substrate of CYP11A1 (module 2) for pregnenolone synthesis (Fig.1f). Duport and colleagues reported that pregnenolone yielded poorly without erg5 deletion, because Erg5 would convert a part of Ergosta-5-ene-ol to Ergosta-5,22-diene-ol, which they reasoned might not be accepted as substrate for CYP11A13. Thus, our strain C alternative might reasonably show lower pregnenolone, therefore testosterone yield.

### Module 2 genes

The products of our first module alternative gene groups (either ergosta-5-ene-ol, or cholesterol) serve as substrate for the second gene module, which comprises CYP11A1 (p450), adrenodoxin, and adrenodoxin reductase (Fig.1f). CYP11A1, also called p450, is a cytochrome monooxygenase that sequentially hydroxylate the C22 and C20 of the cholesterol side chain and eventually cleaves it, leaving behind a keto group at C209,10, known as the side chain cleavage (SCC) activity. The resulting pregnenolone is the key precursor for all steroid hormones in vertebrates. While adrenodoxin serves as a cofactor for CYP11A1, adrenodoxin reductase regenerates reduced adrenodoxin, which is required in the pregnenolone synthesis, say, forward direction9. Ergosta-5-enol, a characteristic intermediate resulting from erg5 deletion (desaturation at C22) and DHCR7 heterologous expression (C7 desaturation), differs from cholesterol, a natural CYP11A1 substrate, in the methyl-group at the C24 position. Duport and colleagues have clearly shown that the side chain of nonconventional ergosta-5-enol is cleaved by CYP11A13. This is further supported by Mast et al., who showed that the side chain binding pocket seem to have allowed structural variations10. However, comparing pregnenolone and peripheral steroid intermediate metabolites yield (as control) between strain A and B may reveal kinetic insights. To construct module 2, cDNA has been taken from bovine origin since Duport et al 1998 have also used cDNA from bovine origin.

### Module 3 genes

HSD3B1, CYP17A1, and HSD17B3 were stated on the KEGG webpage’s “steroid hormone biosynthesis” chart to catalyse the transformation from pregnenolone to testosterone. Therefore, the corresponding human cDNA was used for module 3. The choice to take human cDNA was made in lack of reference publications at the time we had to start the experimental part and order the cDNA, so as a first hit we tried human enzymes since we were aiming to construct a molecule that occurs in humans. Later we saw that Szczebara et al 2003 have successfully used the bovine version of CYP17A1 and HSD3B1 to produce hydrocortisone in S.cerevisae, while Shao et al. had been successful in expressing human HSD17B in P.pastoris and using it for efficient biotransformation of 4-androstene-3,17-dione (AD) to testosterone. The question of which organism to take the enzyme from is an interesting one; yet to optimize our industrial process with regards to choice of enzyme homologues, a follow-up project would be needed.

## Experimental procedure

### Construction of knockout strains

Having a wild type Komagataella phaffii CBS7435 strain and an erg6 knockout strain provided by BOKU, we planned to delete the erg5 gene using PCR to construct a split marker deletion cassette consisting of an antibiotic resistance gene flanked with genomic sequences. Since the erg6 knockout strain already came with an antibiotic resistance, we first had to remove the antibiotic resistance out of the erg6 knockout strain, since we otherwise would have needed more different antibiotic resistance genes than we had at hand. Luckily, we learned that the antibiotic resistance in the erg6 strain had loxP sites, and we succeeded in using Cre recombinase to remove the antibiotic resistance gene. However, we did not succeed in deleting the erg5 gene: Construction of the deletion cassette failed at the stage of fusion PCR that should combine the resistance gene with the genomic flanking sides, and even a thorough analysis did not reveal the reason, since other PCR experiments worked perfectly. As a next step the PCR protocol could have been reviewed, but time did not allow for that. Therefore, we proceeded with the wild type strain and the erg6 knockout as basis for our strain development.

### Plasmid construction

The plasmid modules were constructed as follows: Using Golden Gate cloning, we recombined each gene with a housekeeping P. pastoris promoter (either pGAP, pTEF or pMDH3) and a terminator (either rp12aTT, rpp1bTT, or rps25aTT) and recombined all genes of the same module into a module plasmid (BB3) which contains the genomic homologous recombination sites for P. pastoris. These BB3 plasmids were firstly transformed into E. coli cells for amplification before been transformed into Komagataella phaffii (formerly Pichia pastoris) strains: Module 1a (DHCR7) was inserted in the wild type strain and Module 1b (DHC7 + DHCR24) was inserted in the erg6 knockout strain, while Module 2 and Module 3 were inserted in both of the strains as planned.

### Analytical methods

Quality and quantity of our intermediates and final product were evaluated as follows: The presence of transformed genes was validated using colony PCR. Finally, the successful production of testosterone and its intermediates was validated via HPLC with a known standard. Quantitation of intermediates such as cholesterol and pregnenolone will allow identification of rate limiting steps and hence narrow down possible targets for future trouble shoot or refinements (such as dHeletion, duplication, down or up-regulation of the target). In the future, the product identity could further be verified by NMR and western blot could confirm the presence of the inserted enzymes.

![](https://static.igem.wiki/teams/4700/wiki/images-enginering/a0849a23-bc49-4320-9bc3-8da7b6e91d36.jpg)

FIG.1. a), Experimental strategy of our bioengineering process comprises strain development, integration (transformation) of module 1, 2, and 3 genes, and analytical procedure for characterisation, quantitation and identification for future improvement targets. b)-e): Goal plasmids obtained vis Goldengate serial cloning b) and c): module 1 alternatives (see text for details); d), module 2 genes and e), module 3 gene. f), sterol biochemical synthetic pathway relevant to our process. The end product of module 1 gene (mixture of endogenous [purple] and heterologous [blue] genes) are either ergosterol (endogenous or side product), ergosta-5-monoenol (Duport’s process)3, and cholesterol (Souza’s process)1. The product of second module should be pregnenolone as the different side chains of ergosta-5-monoenol and cholesterol are both cleaved. The ergosterol will also be processed into an epoxide side product due to unsaturated (C24) side chain11.

## References

1. Souza, C. M. et al. A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance. Metab Eng 13, 555–569 (2011).
2. Desmond, E. & Gribaldo, S. Phylogenomics of Sterol Synthesis: Insights into the Origin, Evolution, and Diversity of a Key Eukaryotic Feature. Genome Biol Evol 1, 364–381 (2009).
3. Duport, C., Spagnoli, R., Degryse, E. & Pompon, D. Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast. Nat Biotechnol 16, 186–189 (1998).
4. Gu, Y., Jiao, X., Ye, L. & Yu, H. Metabolic engineering strategies for de novo biosynthesis of sterols and steroids in yeast. Bioresour Bioprocess 8, 110 (2021).
5. Skaggs, B. A. et al. Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene, encoding a second cytochrome P-450 involved in ergosterol biosynthesis. Gene 169, 105–109 (1996).
6. Palermo, L. M., Leak, F. W., Tove, S. & Parks, L. W. Assessment of the essentiality of ERG genes late in ergosterol biosynthesis in Saccharomyces cerevisiae. Curr Genet 32, 93–99 (1997).
7. Abe, F. & Hiraki, T. Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae. Biochimica et Biophysica Acta (BBA) - Biomembranes 1788, 743–752 (2009).
8. Lecain, E., Chenivesse, X., Spagnoli, R. & Pompon, D. Cloning by Metabolic Interference in Yeast and Enzymatic Characterization of *Arabidopsis thaliana* Sterol Δ7-Reductase (∗). Journal of Biological Chemistry 271, 10866–10873 (1996).
9. Simpson, E. R. & Boyd, G. S. The Cholesterol Side-Chain Cleavage System of Bovine Adrenal Cortex. Eur J Biochem 2, 275–285 (1967).
10. Mast, N. et al. Structural Basis for Three-step Sequential Catalysis by the Cholesterol Side Chain Cleavage Enzyme CYP11A1 \* . Journal of Biological Chemistry 286, 5607–5613 (2011).
11. Slominski, A. T. et al. Novel activities of CYP11A1 and their potential physiological significance. J Steroid Biochem Mol Biol 151, 25–37 (2015).

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##### Content

* [Construction of Module 1](#Construction of Module 1)
* [Construction of Module 2](#Construction of Module 2)
* [Construction of Module 3](#Construction of Module 3)
* [Transformation of Pichia pastoris](#Transformation of Pichia pastoris)
* [Cultures for Sterol Determination](#Cultures for Sterol Determination)
* [Extraction](#Extraction)
* [Sterol determination with GC/MS](#Sterol determination with GC/MS)
* [Sterol determination with HPLC](#Sterol determination with HPLC)
* [Protocols](#Protocols)

## Construction of Module 1

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.

## Construction of Module 2

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.

## Construction of Module 3

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.

## Transformation of *Pichia pastoris*

### Wildtype

*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.

### Erg6-KO

*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.

## Cultures for Sterol Determination

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.

## Extraction

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.

### Recovery rates

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.

## Sterol determination with GC/MS

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.

## Sterol determination with HPLC

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.

## Growth

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.

## Erg5-KO

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.

## Protocols

* [Cryostocks of *Escherichia coli*](https://static.igem.wiki/teams/4700/wiki/protocols/cryostocks-of-escherichia-coli.pdf)
* [Culture of *Escherichia coli* for Mini-Prep](https://static.igem.wiki/teams/4700/wiki/protocols/culture-of-escherichia-coli-for-mini-prep.pdf)
* [Determination of DNA Concentration on Nanodrop Device](https://static.igem.wiki/teams/4700/wiki/protocols/determination-of-dna-concentration-on-nanodrop-device.pdf)
* [Extraction of DNA from Electrophoresis Gels](https://static.igem.wiki/teams/4700/wiki/protocols/extraction-of-dna-from-electrophoresis-gels.pdf)
* [Heat Shock Transformation of Chemically Competent *Escherichia coli*](https://static.igem.wiki/teams/4700/wiki/protocols/heat-shock-transformation-of-escherichia-coli.pdf)
* [Measurement of Optical Density of Cultures](https://static.igem.wiki/teams/4700/wiki/protocols/measurement-of-optical-density-of-cultures.pdf)
* [Mini-prep of *Escherichia coli*](https://static.igem.wiki/teams/4700/wiki/protocols/mini-prep-of-escherichia-coli.pdf)
* [Polymerase Chain Reaction – Q5 Polymerase](https://static.igem.wiki/teams/4700/wiki/protocols/polymerase-chain-reaction.pdf)
* [Restriction Enzyme Digest of Plasmids](https://static.igem.wiki/teams/4700/wiki/protocols/restriction-digest.pdf)
* [Small Scale Preparation of Electrocompetent *Pichia pastoris*](https://static.igem.wiki/teams/4700/wiki/protocols/small-scale-preparation-of-electrocompetent-pichia-pastoris.pdf)
* [Transformation of *Pichia pastoris* by Electropolation](https://static.igem.wiki/teams/4700/wiki/protocols/transformation-of-pichia-pastoris-by-electropolation.pdf)
* [Analytic and Preparative Agarose Gel Electrophoresis](https://static.igem.wiki/teams/4700/wiki/protocols/analytic-and-preparative-agarose-gel-electrophoresis.pdf)
* [Media and Antibiotics](https://static.igem.wiki/teams/4700/wiki/protocols/media-and-antibiotics.pdf)
* [gDNA Extraction with Lithium acetate](https://static.igem.wiki/teams/4700/wiki/protocols/gdna-extraction-with-lithium-acetate.pdf)

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##### Content

* [Sterol quantification using HPLC](#Sterol quantification using HPLC)
* [Growth](#Growth)

## Strains

Transformation of Wildtype CBS7435 and Erg6-KO resulted in strains of both with Module 1, Module 1 and 2 and Module 1, 2 and 3. Only the last ones were of further interest for us. Success of Transformation is somewhat plausible, since they grew on media containing NTC, ZEO and HYG simultaneously for a long time, which should not be possible with linearised plasmids Morphology is also consistent with Pichia. However, no colony PCR was conducted and therefore no molecular biological evidence exists to confirm presence. No Testosterone or Pregenolone acetate could be confirmed in our samples.

## Sterol quantification using HPLC

HPLC analysis could not ascertain the presence of Testosterone or Pregnenolone acetate.

![Scheme of Lipid Membrane](https://static.igem.wiki/teams/4700/wiki/results/hplc-analysis-strains.png)
**Figure X:** Relevant area of chromatogram of unmodified wildtype, modified Erg6-KO and modified wildtype. Coloured areas show elution times for Testosterone (red), Pregnenolone acetate (blue), Ergosterol (lime) and Cholestrol (violet), as determined with reference materials.

As the figure shows, neither Testosterone nor Pregnenolone acetate could be found. However, the Erg6-KO shows comparatively little amounts of Ergosterol, meaning the plan to redirect the entire flux into the Testosterone pathway by blocking the Ergosterol pathway is not without merit. In the modified Wildtype a Peak in the Area of cholesterol appears. However, the retention time is slightly different, and the Wildtype shouldn’t be able to produce cholesterol.

## Sterol characterization using GCMS

In both wildtype (ERG6+) DHCR7+module2+module3+ (WT123 denoted in selected-ion monitoring [SIM] chromatogram), wt control strains (WTc), Erg6ΔDHCR7+DHCR24+module2+module3+ (delta6III) strains and Erg6ΔDHCR7-DHCR24-module2-module3- (delta6c) strains, we could not find traces of Testosterone (Fig2a and b), suggesting that our process, so far, is insufficient for testosterone de novo synthesis. We then asked if our strains successfully synthesized pregnenolone. Peaks were present in all of our strains (WTc, WT123, delta6III and delta6c), resembling the retention time of that of pregnenolone (Fig,2c and d). However, when sample SIM chromatograms are compared with that of the pregnenolone acetate standard (Fig2.e and f), only delta6c (unmodified Erg6 deleted) showed peaks with retention time (t=19.782; 20.534) similar to that of pregnenolone acetate (t=19.679). Because Erg6 is a methyltransferase, and many of the enzymes in steroid synthesis pathways are known to be tolerate to similar molecules, deletion of Erg6 may result in characteristic metabolic intermediates. However, this is not observed in delta6III samples (Erg6ΔDHCR7+DHCR24+module2+module3+). Thus, we conclude that our process is unable to synthesize pregnenolone.

Next, we looked at if our strains were able to synthesize cholesterol. Interestingly all strain samples show peaks at the retention time of the cholesterol standard (Fig.2g and h), regardless of the strain (presence of cholesterol synthesis required enzymes). Notably, delta6III might be able to produce a significant amount of cholesterol compared to other strains (peak height: on the scale of 2×10^4), judged by the peak form and relative quantity (about 10% of the standard, not that the y-axis scale is different for different strains in the SIM chromatogram). This suggested that some of our Erg6ΔDHCR7+DHCR24+module2+module3+ (delta6III) strains might be able to synthesize cholesterol successfully. Importantly, further validation processes and investigation should either confirm our primary results. Finally, we characterised the ergosterol production in each of our strains, we found a significant disturbance in delta6III strains (Fig.2j). This is consistent with the function of Erg6. However, the ergosterol peak intensity is heterogenous among delta6c (control) samples, and one replicate showed considerably high ergosterol(Fig.2j). Further analytical steps should investigate is this is due to biological heterogeneity or errors in analytical procedures. In WT samples (123+ and control cells), ergosterol peak intensities are both homogenous in form, and the variance of the peak height (unfortunately, the Area under the peak is not available) is in magnitude of 10^1-10^2(Fig.2i).

Unfortunately, we could not get the designed PCR primers on time. Thus, we could not verify if the gene modules are successfully integrated into the P. pastoris genome and furthermore, determine whether the dysfunction of module 2 and 3 enzymes are due to disturbances on genetic or biochemical levels. From our GS-MS data, we are able to conclude prematurely that some of our delta3III(Erg6ΔDHCR7+DHCR24+module2+module3+) cells are able to produce cholesterol. Though, further analytical procedures and evidences are required for confirmation. For example, a colony PCR test could inform us if the genetic materials are correctly integrated into the yeast’s genome; and biochemical tests, such as westernblot could further provide us valuable information of the end-point expression levels of our desired product, hence further identify the key rate limiting and/or non-functional point.

![Results of our GCMS](https://static.igem.wiki/teams/4700/wiki/gcms-results.jpg)
**Figure X:**

## Growth

The following diagram compares the growth of the produced mutant with steroid hormone pathway with similar strain lacking said pathway.

![Scheme of Lipid Membrane](https://static.igem.wiki/teams/4700/wiki/results/growth-curves.png)
Growth curves of different yeast strains with and without our testosterone pathway

Unsurprisingly, the unmodified wildtype with its normal membrane lipid pathways shows the best growth. However, the growth of the other strains is surprising. First, the unmodified ΔErg6 strain shows the worst growth, significantly lower than the modified ΔErg6 strain. This is counterintuitive since one would assume that the added expression of 6 transgenic proteins and stress from xenobiotic cholesterol and steroid hormones slow growth. A mix up of modified and unmodified ΔErg6 strains was ruled out by growth assays in YPD with and without antibiotics. Another surprise is the very similar growth of both modified strains. Again, one would assume that a strain with an unmolested membrane lipid metabolism grows better than one where a key enzyme was knocked out. These unusual findings point toward some unrecognized experimental error and demand repeat of experiment with fresh pre cultures.

## What to do now?

Current data shows clearly that our strains are unable to produce Testosterone. The question now is, what can be learned and what are possible ways forward. Some possibilities.

* **The strains do not contain our plasmids**. This can be ascertained using simple colony PCR. This would elucidate whether the problem is faulty genes or an unfeasible pathway.
* **The plasmids were damaged during simultaneous integration**This could be possible through homologous recombination with the homologous promotor and terminator sites. This could also be determined through colony PCR and if true possibly remedied by sequential transformation.
* **The strain produces testosterone but very small amounts**Cells used for extraction were cultivated for only 24-36h before harvest. Together with the rather weak constitutional promotors this could mean that just not a lot of testosterone is produced, less than is lost through chemical/biochemical degradation. One of the possibilities is therefore a redesign of the used regulatory elements, a swich to stronger promoters, or methanol induced promotors. These would allow a biomass accumulation before production is turned on.

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