Team:SDU-Denmark/Experiments
Experiments
Abstract
On this page you can read more about the experiments we carried out throughout our project for the production of psilocybin.
Psilocybin is naturally produced by a family of mushrooms, called Psilocybe. This biosynthesis involves four key enzymes, which in five steps convert L-tryptophan into psilocybin.[1] Here L-tryptophan decarboxylase (PsiD) catalyzes the decarboxylation of L-tryptophan to tryptamine. Tryptamine is hydroxylated into 4-hydroxytryptamine by 4-monooxygenase (PsiH). 4-hydroxytryptamine kinase (PsiK) phosphorylates 4-hydroxytryptamine thereby forming nor-baeocystin. Psilocybin synthase (PsiM) catalyzes the transfer of a methyl to the amino group of norbaecystin, which results in the intermediate baeocystin, and a subsequent methyl transfer to the secondary amin of baeocystin, producing psilocybin psilocybin.[1]
For this project, the four genes from Psilocybe cubensis are used as all the enzymes have previously been expressed successfully in eukaryotic organisms as for instance Saccharomyces cerevisiae. Here it must be noticed that it is only PsiD, PsiK, and PsiM which have successfully been expressed in prokaryotes in previous studies.[2] This approach makes the psilocybin production dependent on the expensive precursor 4-hydroxy indole but avoids PsiH which is extremely difficult to express in prokaryotes.[2]
In order to create the de novo synthesis system of psilocybin in E. coli, our experimental approach is based on multiple research papers as well as the work of the Sydney iGEM team of 2019.[3]
The enzymatic pathway for the biosynthesis of psilocybin from substrates such as tryptophan, tryptamine or 4-hydroxytryptamine, was first characterized by (1).[1] The year after Hoefgen et al. 2018 succeeded in the production of psilocybin in a heterologous system.[4] In 2020 further work was conducted with the expression of the PsiD, PsiK, PsiM, and PsiH eukaryotic cells, in this case, Saccharomyces cerevisiae, which was successful but with a concerning 51% of psilocybin being degraded into psilocin passively within the yeast.[5]
The psilocybin producing system
In order to introduce the de novo synthesis system into a proaryotic host, E. coli is used as a production strain. Using E. coli enables a broad and easy biotechnical modeling and cloning due to its characteristics. Implementing the entire system in prokaryotes has never been done successfully before. This is due to PsiH being a cytochrome P450 monooxygenase. These enzymes are usually anchored to the endoplasmic reticulum by a large proportion of their N-terminals in eukaryotic cells, which is also the case for PsiH.[6] Here they catalyze hydroxylation in cooperation with specific reductases such as the NADPH-cytochrome P450 reductase (CPR) that delivers the electrons necessary for the reactions. Like PsiH, CPR is anchored to the endoplasmic reticulum by its N-terminal. Due to the hydrophobic nature of the N-terminals, CPR and PsiH will most likely result in misfolded proteins when expressed in bacteria, as they lack an endoplasmic reticulum.
Modification of the N-terminals and expression of GroEL/ES
Firstly, different kinds of modifications of the enzyme’s N-terminals have been created, to produce more hydrophilic proteins which are soluble in the cytosol of the bacterium. Secondly, previous studies have shown that the overexpression of the native chaperone GroEL/ES is beneficial concerning the expression of P450 monooxygenases. Taking this approach GroEL/ES is overexpressed by co-expressing it in E. coli along with CPR and PsiH to ensure correct folding and optimal catalytic activity.In relation to the N-terminals, there is currently no standard procedure, and it is mostly based on a trial-and-error approach. [7]
In this project, N-terminal variants demonstrating promising results in other studies are used as well as different modifications such as point mutations, deletions, and - most importantly - substitutions of entire N-terminals from other proteins, to make chimeric enzymes. [7] The modified PsiH and CPR enzymes were analyzed in silico by the software RoseTTAFold which uses deep learning to predict how the introduction of the different modifications affects the structure of the enzymes. Model[8]
CPR showed to be very tricky as well because the original one from Psilocybe cubensis (P. cubensis) turned out to consist of several transmembrane segments, thereby making it very difficult to modify using the same technique as described above. Therefore, we have used an orthologue from the other mushroom Phanerochaete chrysosporium, which has a transmembrane domain in the N-terminal only that is much easier to handle using the same approaches as for PsiH.
Point mutations: We decided to replace phenylalanine with alanine because removing big aromatic amino acids and insert alanine instead of has been done with great success before.[9][10] This modification has only been applied on PsiH.
Deletions: For both PsiH and CPR, the signal peptide defined on UniProt was removed as they constitute the only hydrophobic transmembrane domain.[10]
Substitutions: This approach requires that the entire segment of the N-terminal can be identified as we don’t want to affect the catalytic domains. In terms of P450, this is quite easy as it only demands that one determine the proline rich region near the N-terminal, which in our case is PPGPP.[7] For CPR, the end of the N-terminal region was defined by range of the signal peptide.[11] The sequence before these segments can be replaced with N-terminals from other proteins. For PsiH these have been taken from the studies of Ichinose and Wariishi in which lots of different N-terminals from other cytochrome P450s were found to enhance solubility.[7],[12] Some of these have been used for CPR as well, although a few sequences of new N-terminals from water soluble cytochrome b5 reductases have been found using UniProt.
For this project, the four genes from Psilocybe cubensis are used as all the enzymes have previously been expressed successfully in eukaryotic organisms as for instance Saccharomyces cerevisiae. Here it must be noticed that it is only PsiD, PsiK, and PsiM which have successfully been expressed in prokaryotes in previous studies.[2] This approach makes the psilocybin production dependent on the expensive precursor 4-hydroxy indole but avoids PsiH which is extremely difficult to express in prokaryotes.[2]
In order to create the de novo synthesis system of psilocybin in E. coli, our experimental approach is based on multiple research papers as well as the work of the Sydney iGEM team of 2019.[3]
The enzymatic pathway for the biosynthesis of psilocybin from substrates such as tryptophan, tryptamine or 4-hydroxytryptamine, was first characterized by (1).[1] The year after Hoefgen et al. 2018 succeeded in the production of psilocybin in a heterologous system.[4] In 2020 further work was conducted with the expression of the PsiD, PsiK, PsiM, and PsiH eukaryotic cells, in this case, Saccharomyces cerevisiae, which was successful but with a concerning 51% of psilocybin being degraded into psilocin passively within the yeast.[5]
The psilocybin producing system
In order to introduce the de novo synthesis system into a proaryotic host, E. coli is used as a production strain. Using E. coli enables a broad and easy biotechnical modeling and cloning due to its characteristics. Implementing the entire system in prokaryotes has never been done successfully before. This is due to PsiH being a cytochrome P450 monooxygenase. These enzymes are usually anchored to the endoplasmic reticulum by a large proportion of their N-terminals in eukaryotic cells, which is also the case for PsiH.[6] Here they catalyze hydroxylation in cooperation with specific reductases such as the NADPH-cytochrome P450 reductase (CPR) that delivers the electrons necessary for the reactions. Like PsiH, CPR is anchored to the endoplasmic reticulum by its N-terminal. Due to the hydrophobic nature of the N-terminals, CPR and PsiH will most likely result in misfolded proteins when expressed in bacteria, as they lack an endoplasmic reticulum.
Modification of the N-terminals and expression of GroEL/ES
Firstly, different kinds of modifications of the enzyme’s N-terminals have been created, to produce more hydrophilic proteins which are soluble in the cytosol of the bacterium. Secondly, previous studies have shown that the overexpression of the native chaperone GroEL/ES is beneficial concerning the expression of P450 monooxygenases. Taking this approach GroEL/ES is overexpressed by co-expressing it in E. coli along with CPR and PsiH to ensure correct folding and optimal catalytic activity.In relation to the N-terminals, there is currently no standard procedure, and it is mostly based on a trial-and-error approach. [7]
In this project, N-terminal variants demonstrating promising results in other studies are used as well as different modifications such as point mutations, deletions, and - most importantly - substitutions of entire N-terminals from other proteins, to make chimeric enzymes. [7] The modified PsiH and CPR enzymes were analyzed in silico by the software RoseTTAFold which uses deep learning to predict how the introduction of the different modifications affects the structure of the enzymes. Model[8]
CPR showed to be very tricky as well because the original one from Psilocybe cubensis (P. cubensis) turned out to consist of several transmembrane segments, thereby making it very difficult to modify using the same technique as described above. Therefore, we have used an orthologue from the other mushroom Phanerochaete chrysosporium, which has a transmembrane domain in the N-terminal only that is much easier to handle using the same approaches as for PsiH.
Point mutations: We decided to replace phenylalanine with alanine because removing big aromatic amino acids and insert alanine instead of has been done with great success before.[9][10] This modification has only been applied on PsiH.
Deletions: For both PsiH and CPR, the signal peptide defined on UniProt was removed as they constitute the only hydrophobic transmembrane domain.[10]
Substitutions: This approach requires that the entire segment of the N-terminal can be identified as we don’t want to affect the catalytic domains. In terms of P450, this is quite easy as it only demands that one determine the proline rich region near the N-terminal, which in our case is PPGPP.[7] For CPR, the end of the N-terminal region was defined by range of the signal peptide.[11] The sequence before these segments can be replaced with N-terminals from other proteins. For PsiH these have been taken from the studies of Ichinose and Wariishi in which lots of different N-terminals from other cytochrome P450s were found to enhance solubility.[7],[12] Some of these have been used for CPR as well, although a few sequences of new N-terminals from water soluble cytochrome b5 reductases have been found using UniProt.
Inducible promoter systems are used to regulate the protein expression from the psiH, psiDKM, CPR, and groEL/ES genes. There are multiple advantages of using inducible promoters rather than constitutively active ones. Constitutive expression of proteins will often slow the growth of bacteria, as it is energy-consuming and products may be toxic for cells, and thus it is of in-terest that the production does not begin until the exponential growth phase. In the project, two different inducible promoter systems will be used. The aim of this is to be able to express the chaperone GroEL/ES prior to the expression of PsiH, so that the chaperone will be ready to help PsiH fold correctly.[13]
One of the inducible promoter systems used is the pBad/araC system. The repressor protein AraC regulates the transcription of genes controlled by the pBad promoter. In the presence of the monosaccharide L-arabinose, AraC activates the transcription while it actively represses the transcription in the absence of L-arabinose. Thus L-arabinose can be used to induce the tran-scription as desired of GroEL/ES, which is controlled by the pBad/araC promoter.[14]
The other system used is the T7 Lac promoter system. Only the bacteriophage T7-polymerase can initiate transcription from the T7 promoter. In the protein expression strain used (E. coli, ER2566), the T7-polymerase gene is included in the genome, and its expression is controlled by a Lac-promoter and an associated operator site. The T7-promoter controls the genes of interest which are also associated with the lac-operator site. The repressor protein LacI binds to this operator site, and thus inhibits the expression of both the T7-polymerase and the genes of interest. In the presence of the inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG), the LacI repressor protein will let go of the operator site, the T7-promoter will be expressed and thus be able to initiate transcription from the genes of interest. Thus, IPTG can be used to induce the expression of PsiH, PsiDKM, and CPR, when these genes are controlled by a T7-promoter (2).
One of the inducible promoter systems used is the pBad/araC system. The repressor protein AraC regulates the transcription of genes controlled by the pBad promoter. In the presence of the monosaccharide L-arabinose, AraC activates the transcription while it actively represses the transcription in the absence of L-arabinose. Thus L-arabinose can be used to induce the tran-scription as desired of GroEL/ES, which is controlled by the pBad/araC promoter.[14]
The other system used is the T7 Lac promoter system. Only the bacteriophage T7-polymerase can initiate transcription from the T7 promoter. In the protein expression strain used (E. coli, ER2566), the T7-polymerase gene is included in the genome, and its expression is controlled by a Lac-promoter and an associated operator site. The T7-promoter controls the genes of interest which are also associated with the lac-operator site. The repressor protein LacI binds to this operator site, and thus inhibits the expression of both the T7-polymerase and the genes of interest. In the presence of the inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG), the LacI repressor protein will let go of the operator site, the T7-promoter will be expressed and thus be able to initiate transcription from the genes of interest. Thus, IPTG can be used to induce the expression of PsiH, PsiDKM, and CPR, when these genes are controlled by a T7-promoter (2).
For the construction of a plasmid backbone, a resistance cassette carrying re-sistance genes for either, kanamycin-, ampicillin- or chloramphenicol is needed. The use of different antibiotic-resistance cassettes in each plasmid is necessary to ultimately be able to test different combinations of PsiDKM, PsiH, GroEL/ES, and CPR in the same bacteria strain.
For the assembly of an inducible T7 LacI promoter system, LacI and a medium constitutive promotor should be inserted into the respective plasmid backbone. This can be achieved by using the standard iGEM restriction enzyme system: EcoRI, XbaI, SpeI, and PstI. Starting with the J61002 backbone carrying ampicillin resistance and containing a medium constitutive promoter (BBa_J23106), it is possible to insert the LacI gene (BBa number). (Figur) Afterward, the medium constitutive promoter and LacI can be moved together to a kanamycin resistance carrying backbone (pSB1K3) or a chloramphenicol carrying backbone (pSB1C3) as shown in figure. After transformation of ligation products into E. coli Top10, the insert length can be verified by colony-PCR, and the resulting plasmids will now be ready for insertion of biobricks (PsiDKM, PsiH, or CPR).
For GroEL/ES, the plasmid pSB1C3, containing araC and the inducible pBad/araC promoter system (BBa_I0500), is used.
SOPs used: SOP6, SOP7, SOP8, SOP9, SOP12, SOP13
For the assembly of an inducible T7 LacI promoter system, LacI and a medium constitutive promotor should be inserted into the respective plasmid backbone. This can be achieved by using the standard iGEM restriction enzyme system: EcoRI, XbaI, SpeI, and PstI. Starting with the J61002 backbone carrying ampicillin resistance and containing a medium constitutive promoter (BBa_J23106), it is possible to insert the LacI gene (BBa number). (Figur) Afterward, the medium constitutive promoter and LacI can be moved together to a kanamycin resistance carrying backbone (pSB1K3) or a chloramphenicol carrying backbone (pSB1C3) as shown in figure. After transformation of ligation products into E. coli Top10, the insert length can be verified by colony-PCR, and the resulting plasmids will now be ready for insertion of biobricks (PsiDKM, PsiH, or CPR).
For GroEL/ES, the plasmid pSB1C3, containing araC and the inducible pBad/araC promoter system (BBa_I0500), is used.
SOPs used: SOP6, SOP7, SOP8, SOP9, SOP12, SOP13
Creation of single, double, and triple transformants
To create double and triple transformants, the different plasmids are first individually trans-formed into E. coli Top10 and afterward into E. coli ER2566. This two-step approach was used as the Top10 strain distinguishes itself by being good for cloning while ER2566 is good for protein expression. The ligation product-based transformation into Top10 is quality controlled by colony PCR. Here it is possible to distinguish between successful and relegation-based transformations.
The finished ER2566 single transformants were then included in transcription and translation control experiments and in addition sent to Eurofins for sequencing. Following this, triple transformants were created in a stepwise approach, by transforming one plasmid into the bacteria at a time. The ER2566 triple transformants were quality controlled by primer specific colony PCR. Taking these triple transformants further the production strain is changed to two different efflux pump knockout strains. Succeeding, the whole de novo synthesis system can be introduced into the photoautotroph R. sulfidophilum Hardware.
SOPs used: SOP9, SOP14, SOP17
To create double and triple transformants, the different plasmids are first individually trans-formed into E. coli Top10 and afterward into E. coli ER2566. This two-step approach was used as the Top10 strain distinguishes itself by being good for cloning while ER2566 is good for protein expression. The ligation product-based transformation into Top10 is quality controlled by colony PCR. Here it is possible to distinguish between successful and relegation-based transformations.
The finished ER2566 single transformants were then included in transcription and translation control experiments and in addition sent to Eurofins for sequencing. Following this, triple transformants were created in a stepwise approach, by transforming one plasmid into the bacteria at a time. The ER2566 triple transformants were quality controlled by primer specific colony PCR. Taking these triple transformants further the production strain is changed to two different efflux pump knockout strains. Succeeding, the whole de novo synthesis system can be introduced into the photoautotroph R. sulfidophilum Hardware.
Agarose gel with pSB1K3-LacI-T7lac-PsiDKM: Bands ~6000 indicate successful assembly of pSB1K3-LacI-T7lac-PsiDKM. The lanes correspond to the different colonies obtained from the transformation of pSB1K3-LacI-T7lac-PsiDKM.
SOPs used: SOP9, SOP14, SOP17
Confirmation of enzymatic activity is crucial for optimal production of psilocybin. Adding IPTG to the LacI system will induce the gene expression of the four enzymes PsiD, PsiK, PsiM, PsiH, and CPR. This can be confirmed on a transcriptional level using quantitative PCR (qPCR). By using qPCR, it is possible to confirm the activity of both the enzymes and the promotor system.
Furthermore, the gene expression of the enzymes can be confirmed on a translational level using SDS-PAGE, in which an increased expression can be seen on the SDS-Page gel as an intensified band, compared to a sample that has not been induced with IPTG.
Furthermore, the gene expression of the enzymes can be confirmed on a translational level using SDS-PAGE, in which an increased expression can be seen on the SDS-Page gel as an intensified band, compared to a sample that has not been induced with IPTG.
Approach
Our main point of interest is the assembly of the three enzymes PsiD, PsiK, and PsiM, which previously have been shown to produce psilocybin from the substrate 4-hydroxy indole.
Assembly of PsiDKM
The enzymes PsiD, PsiK, and PsiM biobricks were designed as two separate bricks - one containing PsiD and PsiK and one containing PsiM. The design of a two biobrick systems was necessary due to the ability for IDT to only synthesize 3000bp at a time. For amplification of the two bricks PCR can be used, using the Phusion High-Fidelity PCR Master Mix. For the amplification of the bricks primers with complementary overhangs were used. The amplified bricks were separated with gelelectrophoresis followed by purification from gel.
For the assembly of PsiDKM the two bricks were merged and amplified using overhang PCR and the Phusion High-Fidelity PCR Master Mix. Gel electrophoresis confirms the assembly of PsiDKM, due to the knowledge of an expected length contained from Snap Gene and afterward purified from gel. Furthermore, the assembly of PsiDKM can be confirmed by sequencing.
SOPs used: SOP6, SOP8, SOP10, SOP17
Assembly of plasmid backbone and PsiDKM
For the assembly of the plasmid backbone containing kanamycin resistance (pSB1K3-LacI) and PsiDKM (BBa_K4059037), the plasmid backbone should be digested using the restriction enzymes SpeI and PstI. The digestion can be confirmed using gelelectrophoresis and afterward purified from gel. Furthermore, PsiDKM should be digested with the restriction enzymes XbaI and PstI and afterward confirmed by gelelectrophoresis followed by purification from gel.
For the assembly of pSB1K3-LacI and PsiDKM a ligation mixture of digested pSB1K3-LacI and PsiDKM should be prepared followed by transformation into E. coli Top10 strain. Colony PCR can be used to confirm the assembly. The plasmid DNA from the transformed cells can be purified using miniprep resulting in pSB1K3-LacI-T7lac-PsiDKM. For the confirmation of pSB1K3-LacI-T7lac-PsiDKM sequencing was used.
pSB1K3-LacI-T7lac-PsiDKM should be transformed into E. Coli ER2566 strain. To analyze the expression of PsiDKM after inducing with IPTG, RNA purification followed by Real-Time Quantitative PCR (qPCR) was performed. The results of Real-Time qPCR were used to confirm the activity of PsiDKM.
SOPs used: SOP6, SOP7, SOP8, SOP9, SOP12, SOP13, SOP14, SOP17, SOP18, SOP24
Our main point of interest is the assembly of the three enzymes PsiD, PsiK, and PsiM, which previously have been shown to produce psilocybin from the substrate 4-hydroxy indole.
Assembly of PsiDKM
The enzymes PsiD, PsiK, and PsiM biobricks were designed as two separate bricks - one containing PsiD and PsiK and one containing PsiM. The design of a two biobrick systems was necessary due to the ability for IDT to only synthesize 3000bp at a time. For amplification of the two bricks PCR can be used, using the Phusion High-Fidelity PCR Master Mix. For the amplification of the bricks primers with complementary overhangs were used. The amplified bricks were separated with gelelectrophoresis followed by purification from gel.
For the assembly of PsiDKM the two bricks were merged and amplified using overhang PCR and the Phusion High-Fidelity PCR Master Mix. Gel electrophoresis confirms the assembly of PsiDKM, due to the knowledge of an expected length contained from Snap Gene and afterward purified from gel. Furthermore, the assembly of PsiDKM can be confirmed by sequencing.
SOPs used: SOP6, SOP8, SOP10, SOP17
Assembly of plasmid backbone and PsiDKM
For the assembly of the plasmid backbone containing kanamycin resistance (pSB1K3-LacI) and PsiDKM (BBa_K4059037), the plasmid backbone should be digested using the restriction enzymes SpeI and PstI. The digestion can be confirmed using gelelectrophoresis and afterward purified from gel. Furthermore, PsiDKM should be digested with the restriction enzymes XbaI and PstI and afterward confirmed by gelelectrophoresis followed by purification from gel.
Assembly of pSB1K3-LacI and PsiDKM:: The expected results for gel electrophoresis for backbone, pSB1K3-LacI, and PsiDKM
For the assembly of pSB1K3-LacI and PsiDKM a ligation mixture of digested pSB1K3-LacI and PsiDKM should be prepared followed by transformation into E. coli Top10 strain. Colony PCR can be used to confirm the assembly. The plasmid DNA from the transformed cells can be purified using miniprep resulting in pSB1K3-LacI-T7lac-PsiDKM. For the confirmation of pSB1K3-LacI-T7lac-PsiDKM sequencing was used.
Assembled pSB1K3-LacI-T7lac-PsiDKM: The expected results for gel electrophoresis for pSB1K3-LacI-T7lac-PsiDKM.
pSB1K3-LacI-T7lac-PsiDKM should be transformed into E. Coli ER2566 strain. To analyze the expression of PsiDKM after inducing with IPTG, RNA purification followed by Real-Time Quantitative PCR (qPCR) was performed. The results of Real-Time qPCR were used to confirm the activity of PsiDKM.
SOPs used: SOP6, SOP7, SOP8, SOP9, SOP12, SOP13, SOP14, SOP17, SOP18, SOP24
Our approach
Wild type PsiH and CPR have hydrophobic N-terminal domains which enable them to be associated with the endoplasmic reticulum membrane in the eukaryotic fungi cell. However, these intracellular membrane structures are not present in the prokaryotic E. coli cell. Due to this the N-terminals must be modified, making them more hydrophilic and thus enabling the expression of the enzyme in the cytosol without altering the 3° structure of the entire enzyme. In the PsiH and CPR biobricks ordered from IDT the Psilocybe cubensis wild type gene sequences have been codon optimized for E. coli. Furthermore, the N-terminal encoding WT gene regions have been deleted.
The different N-terminals were introduced by overhang PCR. This method was used to conveniently produce different CPR and PsiH N-terminal variants with the use of designed over-hang oligoes rather than using complete individual biobricks for each modification. The overhang PCR products were inserted in different backbones and transformed following the general two step transformation approach.
The successful single ER2566 transformants of CPR and PsiH N-terminal variants, and Gro-EL/ES were moved to different antibiotic resistance carrying backbones, enabling the creation of double and lastly triple transformant systems. The three plasmids were introduced stepwise into ER2566. Lastly, the triple systems were included in production experiments, aiming to produce psilocybin from either 4-Hydroxyindole or Tryptophan, which yield was quantified using liquid chromatography-mass spectrometry.
Wild type PsiH and CPR have hydrophobic N-terminal domains which enable them to be associated with the endoplasmic reticulum membrane in the eukaryotic fungi cell. However, these intracellular membrane structures are not present in the prokaryotic E. coli cell. Due to this the N-terminals must be modified, making them more hydrophilic and thus enabling the expression of the enzyme in the cytosol without altering the 3° structure of the entire enzyme. In the PsiH and CPR biobricks ordered from IDT the Psilocybe cubensis wild type gene sequences have been codon optimized for E. coli. Furthermore, the N-terminal encoding WT gene regions have been deleted.
The different N-terminals were introduced by overhang PCR. This method was used to conveniently produce different CPR and PsiH N-terminal variants with the use of designed over-hang oligoes rather than using complete individual biobricks for each modification. The overhang PCR products were inserted in different backbones and transformed following the general two step transformation approach.
The successful single ER2566 transformants of CPR and PsiH N-terminal variants, and Gro-EL/ES were moved to different antibiotic resistance carrying backbones, enabling the creation of double and lastly triple transformant systems. The three plasmids were introduced stepwise into ER2566. Lastly, the triple systems were included in production experiments, aiming to produce psilocybin from either 4-Hydroxyindole or Tryptophan, which yield was quantified using liquid chromatography-mass spectrometry.
Creation of N-terminal variants of PsiH and CPR
To be able to conveniently create numerous different N-terminal modified PsiH and CPR enzymes, overhang PCR is used. By this approach, it is possible to substitute the N-terminals. The standard PsiH and CPR biobricks from IDT were resuspended. Following this step, two PCR reactions were run to introduce the new N-terminals following. Here it must be noted that as the PsiH and CPR biobricks do not contain a T7 promotor and an RBS site, two overhang introducing PCR reactions had to be run. By using a forward primer specific for the T7lac promoter and an N-terminal variant-specific reverse overhang primer, a 5’-overhang could be introduced. For the PsiH or CPR biobrick a normal reverse primer together with a variant-specific forward overhang primer was used. This resulted in the creation of a 5’-overhang for the PsiH biobrick. The products of the overhang introducing PCR reactions were quality controlled by gel electrophoresis. The expected length of the different bands was predicted using SnapGene and compared to the experimental data.
SOPs used: SOP16, SOP10, SOP8
The bands confirming a successful introduction of overhangs were prepared for PCR reaction number two with the aim to assemble overhang DNA 1 and 2. Following this step, the N-terminal modified CPR or PsiH products are inserted in different antibiotic resistance carrying backbones.
SOPs used: SOP6, SOP7, SOP12, SOP13
To be able to conveniently create numerous different N-terminal modified PsiH and CPR enzymes, overhang PCR is used. By this approach, it is possible to substitute the N-terminals. The standard PsiH and CPR biobricks from IDT were resuspended. Following this step, two PCR reactions were run to introduce the new N-terminals following. Here it must be noted that as the PsiH and CPR biobricks do not contain a T7 promotor and an RBS site, two overhang introducing PCR reactions had to be run. By using a forward primer specific for the T7lac promoter and an N-terminal variant-specific reverse overhang primer, a 5’-overhang could be introduced. For the PsiH or CPR biobrick a normal reverse primer together with a variant-specific forward overhang primer was used. This resulted in the creation of a 5’-overhang for the PsiH biobrick. The products of the overhang introducing PCR reactions were quality controlled by gel electrophoresis. The expected length of the different bands was predicted using SnapGene and compared to the experimental data.
SOPs used: SOP16, SOP10, SOP8
The bands confirming a successful introduction of overhangs were prepared for PCR reaction number two with the aim to assemble overhang DNA 1 and 2. Following this step, the N-terminal modified CPR or PsiH products are inserted in different antibiotic resistance carrying backbones.
SOPs used: SOP6, SOP7, SOP12, SOP13
Our approach - Why transport?
Most heterologous production relies on the lysis of cells to liberate their product. Lysis requires the continuous restart of growth of the production organism, but this is dependent on many resources and adds a workload. Therefore, we decided on a different approach to accommodate the green future.
Our approach relies on the use of transporters to transport psilocybin out of the cell thereby skipping the lysis step in manufacturing. This allows for continuous production of psilocybin in a bioreactor ,where only the media would need to be changed and no lysis of the cells would be necessary.
Furthermore, transporting the product out of the cell hinders a possible buildup of psilocybin that could potentially slow production. To actualize our vision of continuous transport out of the cell we decided upon two different approaches; one, which includes testing of two P. cubensis theoretical native transporters and second, a native E. coli transporter.
P. cubensis transporters
In literature we found a description of a genetic island containing two genes that were hypothesized as transporters for psilocybin.[1] The genes were named psiT1 and psiT2 and the protein products Major facilitator-type transporter psiT1 (BBa_K4059032) (PsiT1 for short) and Major facilitator-type transporter psiT2 (BBa_K4059033) (PsiT2 for short) respectively. The transporters were still on the theoretical basis wherefore we examined the amino acid sequence in Uniprot. We used Swiss-Model for protein structure homology-modelling and found that Ma-jor facilitator-type transporter psiT1 and Major facilitator-type transporter psiT2 show similarity with known ion coupled transporters. This indicated that the proteins are transporters which drove us to continue with experiments. However, as we did not know how these transporters would be oriented in a membrane of E. coli or even if they would localize to the membrane, we designed our experiments around these questions. We designed a method for investigating if a protein localizes to the membrane, as well as the orientation. This method relies on coupling GFP to the N-terminal and C-terminal of the protein in two different plasmid constructs. The constructs consist of GFP modified from BBa_K125500 from the iGEM registry linked to PsiT1 (BBa_K4059032) and PsiT2 (BBa_K4059033). The linker chosen was BBa_K1486004 from the iGEM registry which was incorporated together with BBa_K125500 in our BBa_K4059034 and BBa_K4059035 parts.
Plasmid design
In designing our biobricks we thought about possible use in future connections with membrane proteins. To assemble our biobricks we chose a backbone BBa_J61002 from the iGEM registry with an ampicillin resistance marker for ease in our transformations. Our primary design was the designing of GFP-constructs that can be coupled to C-terminal of transporters or N-terminal of transporters. The GFP sequence was taken from BBa_K125500 in the iGEM registry. The constructs were designed to include a constitutive promoter BBa_J23100 to ensure contin-uous expression of the inserted proteins (PsiT1 and PsiT2). We then designed N-terminal- and C-terminal GFP coupled biobricks specific sequences to be ligated to our specific transporters by use of non-standard iGEM restriction enzymes NdeI and BamHI as described further in assembly of constructs for microscopy. In order to introduce the specific NdeI restriction site, it was necessary to use a filler for this. We chose the filler 5'-TATACAT prior to the start codon of the transporters for the introduction of this site.
This plasmid shows the GFP-C-terminal construct (BBa_K4059034) coupled to our PsiT2 transporter.
This plasmid shows the GFP-N-terminal construct (BBa_K4059035) coupled to our PsiT2 transporter.
This plasmid shows the GFP-C-terminal construct (BBa_K4059034) coupled to our PsiT1 transporter.
This plasmid shows the GFP-N-terminal construct (BBa_K4059035) coupled to our PsiT1 transporter.
E. coli native transporter
As the P. cubensis native transporter might cause issues with expression or localization to the membrane as was our concern with the PsiH enzyme. As the first approach relies on the use of eukaryotic membrane proteins, we decided to look for possible candidate transporters in E. coli. During extensive literature research we found an article describing the transport of different dyes containing extensive ring systems through the native E. coli transporters; YhjV, YihN and TolC. [15] We also found that a patent was given in 2020 concerning YhjV as a transporter of indole derivatives. [16] As for PsiT1 and 2, we performed protein structure homology-modelling using Swiss-Model, which indicated that YhjV is an antitransporter - and even more intriguing; antitransporter of amino acids. We found this quite interesting as an ideal transport mechanism for our psilocybin producing system would be a scenario in which tryptophan, our start substrate, would be carried inside the cell while psilocybin should be exported. The transporter YhjV is described to be involved in the transport of the dyes with extensive ring systems as the cells express significantly more fluorescence when this is upregulated, and less fluorescence when this is knocked out.[15] Since this transporter was also described in the patent application as a transporter of indole derivatives, we chose to investigate this further.
AcrA E. coli native efflux pump
The intermediates in our pathway all share a common feature of being indoles, wherefore it would be an issue if these are exported from the cell. This can be done through efflux pumps of which we chose to focus on AcrA which is part of the multi-drug efflux pump along with AcrB and TolC. The pump is composed of the components in a 1:2:1: AcrB:AcrA:TolC.[17] This efflux pump has broad substrate specificity but has been described in the patent as a transporter of indole derivatives.[16] As the pump is an efflux pump it is logical to assume that it could therefore export our intermediates from the cell thereby eventually lessening our output of psilocybin. It is further described in the literature that indoles slightly upregulate the expression of AcrA, which could therefore potentially cause issues in our production of psilocybin and a lower yield.[18]
Knockout experiments
Investigating the effect of both YhjV as a possible transporter for our psilocybin and AcrA as an exporter of our intermediates we constructed knockout mutants in E. coli. We constructed the knockouts from strains in the keio collection specifically JW0452 and JW3508. P1 phages are involved in the transfer of the knockout of genes from JW0452 by replacing the AcrA gene with the kanamycin cassette from JW0452 in E. coli. The same procedure was carried out for yhjV from JW3508. We then utilized the ∆yhjV strain for mass-spectrometry to see if this lowered the yield of psilocybin and intermediates as described further
Most heterologous production relies on the lysis of cells to liberate their product. Lysis requires the continuous restart of growth of the production organism, but this is dependent on many resources and adds a workload. Therefore, we decided on a different approach to accommodate the green future.
Our approach relies on the use of transporters to transport psilocybin out of the cell thereby skipping the lysis step in manufacturing. This allows for continuous production of psilocybin in a bioreactor ,where only the media would need to be changed and no lysis of the cells would be necessary.
Furthermore, transporting the product out of the cell hinders a possible buildup of psilocybin that could potentially slow production. To actualize our vision of continuous transport out of the cell we decided upon two different approaches; one, which includes testing of two P. cubensis theoretical native transporters and second, a native E. coli transporter.
P. cubensis transporters
In literature we found a description of a genetic island containing two genes that were hypothesized as transporters for psilocybin.[1] The genes were named psiT1 and psiT2 and the protein products Major facilitator-type transporter psiT1 (BBa_K4059032) (PsiT1 for short) and Major facilitator-type transporter psiT2 (BBa_K4059033) (PsiT2 for short) respectively. The transporters were still on the theoretical basis wherefore we examined the amino acid sequence in Uniprot. We used Swiss-Model for protein structure homology-modelling and found that Ma-jor facilitator-type transporter psiT1 and Major facilitator-type transporter psiT2 show similarity with known ion coupled transporters. This indicated that the proteins are transporters which drove us to continue with experiments. However, as we did not know how these transporters would be oriented in a membrane of E. coli or even if they would localize to the membrane, we designed our experiments around these questions. We designed a method for investigating if a protein localizes to the membrane, as well as the orientation. This method relies on coupling GFP to the N-terminal and C-terminal of the protein in two different plasmid constructs. The constructs consist of GFP modified from BBa_K125500 from the iGEM registry linked to PsiT1 (BBa_K4059032) and PsiT2 (BBa_K4059033). The linker chosen was BBa_K1486004 from the iGEM registry which was incorporated together with BBa_K125500 in our BBa_K4059034 and BBa_K4059035 parts.
Plasmid design
In designing our biobricks we thought about possible use in future connections with membrane proteins. To assemble our biobricks we chose a backbone BBa_J61002 from the iGEM registry with an ampicillin resistance marker for ease in our transformations. Our primary design was the designing of GFP-constructs that can be coupled to C-terminal of transporters or N-terminal of transporters. The GFP sequence was taken from BBa_K125500 in the iGEM registry. The constructs were designed to include a constitutive promoter BBa_J23100 to ensure contin-uous expression of the inserted proteins (PsiT1 and PsiT2). We then designed N-terminal- and C-terminal GFP coupled biobricks specific sequences to be ligated to our specific transporters by use of non-standard iGEM restriction enzymes NdeI and BamHI as described further in assembly of constructs for microscopy. In order to introduce the specific NdeI restriction site, it was necessary to use a filler for this. We chose the filler 5'-TATACAT prior to the start codon of the transporters for the introduction of this site.
This plasmid shows the GFP-C-terminal construct (BBa_K4059034) coupled to our PsiT2 transporter.
This plasmid shows the GFP-N-terminal construct (BBa_K4059035) coupled to our PsiT2 transporter.
This plasmid shows the GFP-C-terminal construct (BBa_K4059034) coupled to our PsiT1 transporter.
This plasmid shows the GFP-N-terminal construct (BBa_K4059035) coupled to our PsiT1 transporter.
E. coli native transporter
As the P. cubensis native transporter might cause issues with expression or localization to the membrane as was our concern with the PsiH enzyme. As the first approach relies on the use of eukaryotic membrane proteins, we decided to look for possible candidate transporters in E. coli. During extensive literature research we found an article describing the transport of different dyes containing extensive ring systems through the native E. coli transporters; YhjV, YihN and TolC. [15] We also found that a patent was given in 2020 concerning YhjV as a transporter of indole derivatives. [16] As for PsiT1 and 2, we performed protein structure homology-modelling using Swiss-Model, which indicated that YhjV is an antitransporter - and even more intriguing; antitransporter of amino acids. We found this quite interesting as an ideal transport mechanism for our psilocybin producing system would be a scenario in which tryptophan, our start substrate, would be carried inside the cell while psilocybin should be exported. The transporter YhjV is described to be involved in the transport of the dyes with extensive ring systems as the cells express significantly more fluorescence when this is upregulated, and less fluorescence when this is knocked out.[15] Since this transporter was also described in the patent application as a transporter of indole derivatives, we chose to investigate this further.
AcrA E. coli native efflux pump
The intermediates in our pathway all share a common feature of being indoles, wherefore it would be an issue if these are exported from the cell. This can be done through efflux pumps of which we chose to focus on AcrA which is part of the multi-drug efflux pump along with AcrB and TolC. The pump is composed of the components in a 1:2:1: AcrB:AcrA:TolC.[17] This efflux pump has broad substrate specificity but has been described in the patent as a transporter of indole derivatives.[16] As the pump is an efflux pump it is logical to assume that it could therefore export our intermediates from the cell thereby eventually lessening our output of psilocybin. It is further described in the literature that indoles slightly upregulate the expression of AcrA, which could therefore potentially cause issues in our production of psilocybin and a lower yield.[18]
Knockout experiments
Investigating the effect of both YhjV as a possible transporter for our psilocybin and AcrA as an exporter of our intermediates we constructed knockout mutants in E. coli. We constructed the knockouts from strains in the keio collection specifically JW0452 and JW3508. P1 phages are involved in the transfer of the knockout of genes from JW0452 by replacing the AcrA gene with the kanamycin cassette from JW0452 in E. coli. The same procedure was carried out for yhjV from JW3508. We then utilized the ∆yhjV strain for mass-spectrometry to see if this lowered the yield of psilocybin and intermediates as described further
The experiments – Transport Assembly
To assemble transporters (BBa_K4059032 and BBa_K4059033) with the GFP-constructs (BBa_K4059034 and BBa_K4059035) the following non-standard restriction enzymes are in-volved with the specified restriction sites: NdeI: 5'-CATATG'-3 and BamHI: 5'-GGATCC-'3.
Assembly of backbones
The N-terminal and C-terminal backbones were designed as two separate biobricks, both con-taining GFP sequence from BBa_K125500 and then each containing either the specific N-terminal or C-terminal. The bricks are amplified using Phusion PCR followed by gel electro-phoresis and purification from gel.
The two biobricks
The GFP-constructs are inserted into our backbone Bba_J61002 with Bba_J23106 through ligation of these two parts. Cutting of both N-terminal and C-terminal and Bba_J61002 should be carried out with Xba1 and Pst1 followed by gel electrophoresis and gel purification. This cutting allows ligation to be carried out between gel purified C-terminal GFP protein and Bba_J61002, and N-terminal GFP protein and Bba_J61002. The cut with XbaI and PstI removes both ribosomal binding site and promoter as this can be introduced later. The assembly can be carried out through ligation of which the products can then be used further for TSB transformation.
SOPs used: SOP6, SOP7, SOP8, SOP11, SOP13, SOP14
Assembly of constructs for microscopy
Once the C-terminal GFP construct (BBa_K4059034) and N-terminal GFP construct (BBa_K4059035) have been successfully assembled through ligation these can then be cut once again. This time the cutting can be done with different restriction enzymes which are not standard enzymes in iGEM. These enzymes are NdeI and BamHI.
The transporters PsiT1 and PsiT2 (Bba_K4059032 and Bba_K4059033) must have the BamHI restriction site introduced through PCR with Phusion in which the two transporters are amplified with both a suffix and prefix specific primer for the introduction of the BamHI restriction site.
N-terminal fusion to GFP construct: The transporter should be amplified using a suffix primer and primer that introduces BamHI site at the N-terminal of the protein. Then both the plasmid with construct and protein of interest are cut with BamHI + SpeI so that they will be compatible and the cutting is verified by gel electrophoresis followed by purification from gel.
C-terminal fusion to GFP construct: The protein of interest should be amplified with a prefix primer and the primer that introduces the BamHI site at the C-terminal, after which both the plasmid with the construct and the protein of interest are cut with NdeI + BamHI and the cutting is verified by gel electrophoresis followed by purification from gel. The ligation products can then be transformed into E. coli and selected based on the ampicillin resistance marker present in Bba_J61002. This is verified by Colony PCR. The expected colony PCR results would be that the amplification with suffix and prefix primer would show the insert of 1145 bp and 1148 bp of N-terminal GFP construct and C-terminal GFP constructs (BBa_K4059035 and BBa_K4059034) in well 1 and well 2 below respectively.
SOPs used: SOP6, SOP8, SOP9
Fluorescence microscopy
Once the GFP has been successfully coupled to N-terminal and C-terminal GFP constructs part of our transporters, the constructs can be used to investigate the orientation of the transporters as well as their ability to be integrated in the membrane. If the transporter was oriented with the terminal coupled to GFP intracellularly, only a halo of florescence occurs as seen schematically below:
If the transporter was oriented in such a way that the GFP would be found extracellularly, no fluorescence will be seen, since GFP cannot fold correctly outside the cell due to different pH.[19]
However, in case of misfolding or incorrectly expressed transporter, this results in a general florescence of the cell, seen as small dots or granulates consisting of aggregated proteins as seen below:
This allows visualization of whether the transporter was integrated into the membrane as fluo-rescence would then be localized to the cell membrane and not generally in the cytosol.
SOPs used: SOP19
Western Blot
Western blot is carried out to investigate potential cleavage of GFP and transporters in the link-er region as this region contains a lot of serine along with glycine to produce a very flexible peptide chain. E. coli is a bacteria-rich in serine proteases.[20] This is carried out with GFP antibody and secondary HRP conjugated antibody. If cleavage had occurred, it could be seen as GFP in its size of 27.4 kDa according to Snapgene and possibly the transporters at their respective sizes. However, it is possible that the cleavage could occur in other places as well. This is shown in the figure below:
If no cleavage had occurred, it would be seen as the full size of the transporters with the GFP-constructs added at 79.2 kDa and 84.3 kDa for PsiT1 and PsiT2. This is shown in the figure below:
SOPs used: SOP22
To assemble transporters (BBa_K4059032 and BBa_K4059033) with the GFP-constructs (BBa_K4059034 and BBa_K4059035) the following non-standard restriction enzymes are in-volved with the specified restriction sites: NdeI: 5'-CATATG'-3 and BamHI: 5'-GGATCC-'3.
Assembly of backbones
The N-terminal and C-terminal backbones were designed as two separate biobricks, both con-taining GFP sequence from BBa_K125500 and then each containing either the specific N-terminal or C-terminal. The bricks are amplified using Phusion PCR followed by gel electro-phoresis and purification from gel.
The two biobricks
The GFP-constructs are inserted into our backbone Bba_J61002 with Bba_J23106 through ligation of these two parts. Cutting of both N-terminal and C-terminal and Bba_J61002 should be carried out with Xba1 and Pst1 followed by gel electrophoresis and gel purification. This cutting allows ligation to be carried out between gel purified C-terminal GFP protein and Bba_J61002, and N-terminal GFP protein and Bba_J61002. The cut with XbaI and PstI removes both ribosomal binding site and promoter as this can be introduced later. The assembly can be carried out through ligation of which the products can then be used further for TSB transformation.
SOPs used: SOP6, SOP7, SOP8, SOP11, SOP13, SOP14
Assembly of constructs for microscopy
Once the C-terminal GFP construct (BBa_K4059034) and N-terminal GFP construct (BBa_K4059035) have been successfully assembled through ligation these can then be cut once again. This time the cutting can be done with different restriction enzymes which are not standard enzymes in iGEM. These enzymes are NdeI and BamHI.
The transporters PsiT1 and PsiT2 (Bba_K4059032 and Bba_K4059033) must have the BamHI restriction site introduced through PCR with Phusion in which the two transporters are amplified with both a suffix and prefix specific primer for the introduction of the BamHI restriction site.
N-terminal fusion to GFP construct: The transporter should be amplified using a suffix primer and primer that introduces BamHI site at the N-terminal of the protein. Then both the plasmid with construct and protein of interest are cut with BamHI + SpeI so that they will be compatible and the cutting is verified by gel electrophoresis followed by purification from gel.
C-terminal fusion to GFP construct: The protein of interest should be amplified with a prefix primer and the primer that introduces the BamHI site at the C-terminal, after which both the plasmid with the construct and the protein of interest are cut with NdeI + BamHI and the cutting is verified by gel electrophoresis followed by purification from gel. The ligation products can then be transformed into E. coli and selected based on the ampicillin resistance marker present in Bba_J61002. This is verified by Colony PCR. The expected colony PCR results would be that the amplification with suffix and prefix primer would show the insert of 1145 bp and 1148 bp of N-terminal GFP construct and C-terminal GFP constructs (BBa_K4059035 and BBa_K4059034) in well 1 and well 2 below respectively.
SOPs used: SOP6, SOP8, SOP9
Fluorescence microscopy
Once the GFP has been successfully coupled to N-terminal and C-terminal GFP constructs part of our transporters, the constructs can be used to investigate the orientation of the transporters as well as their ability to be integrated in the membrane. If the transporter was oriented with the terminal coupled to GFP intracellularly, only a halo of florescence occurs as seen schematically below:
If the transporter was oriented in such a way that the GFP would be found extracellularly, no fluorescence will be seen, since GFP cannot fold correctly outside the cell due to different pH.[19]
However, in case of misfolding or incorrectly expressed transporter, this results in a general florescence of the cell, seen as small dots or granulates consisting of aggregated proteins as seen below:
This allows visualization of whether the transporter was integrated into the membrane as fluo-rescence would then be localized to the cell membrane and not generally in the cytosol.
SOPs used: SOP19
Western Blot
Western blot is carried out to investigate potential cleavage of GFP and transporters in the link-er region as this region contains a lot of serine along with glycine to produce a very flexible peptide chain. E. coli is a bacteria-rich in serine proteases.[20] This is carried out with GFP antibody and secondary HRP conjugated antibody. If cleavage had occurred, it could be seen as GFP in its size of 27.4 kDa according to Snapgene and possibly the transporters at their respective sizes. However, it is possible that the cleavage could occur in other places as well. This is shown in the figure below:
If no cleavage had occurred, it would be seen as the full size of the transporters with the GFP-constructs added at 79.2 kDa and 84.3 kDa for PsiT1 and PsiT2. This is shown in the figure below:
SOPs used: SOP22
The experiments – Knockout
To test the transporters acrA and yhjV’s role in the efflux of intermediates and of psilocybin respectively, a knockout of the transporters will be performed. P1 phages are obtained and used for the making of phage lysate. Phage lysate is made of the two knockout mutants from the keio collection, acrA, and yhjV. When the lysate is transduced into E. coli, genes for acrA or yhjV are replaced with an FRT-flanked kanamycin resistance cassette.[21] The transduced bacteria are selected for kanamycin to test if the cassette has been inserted correctly. Once the kanamycin cassette is introduced, it needs to be excised. Here a plasmid containing pCP20 and an ampicillin marker is introduced into the bacteria. pCP20 is an FLP recombinase that mediates the recombination between the FRT sites flanking the kanamycin cassette. The recombinase is temperature-sensitive and must be grown at temperatures below 30oC to introduce the plasmid into the cell. To complete the knockout the recombinase is cured at 42℃, removing it from the bacteria.[21] By performing a colony PCR over the created knockout mutants, the length should indicate a smaller fragment size than the WT ER2566.
SOPs used: SOP20
To test the transporters acrA and yhjV’s role in the efflux of intermediates and of psilocybin respectively, a knockout of the transporters will be performed. P1 phages are obtained and used for the making of phage lysate. Phage lysate is made of the two knockout mutants from the keio collection, acrA, and yhjV. When the lysate is transduced into E. coli, genes for acrA or yhjV are replaced with an FRT-flanked kanamycin resistance cassette.[21] The transduced bacteria are selected for kanamycin to test if the cassette has been inserted correctly. Once the kanamycin cassette is introduced, it needs to be excised. Here a plasmid containing pCP20 and an ampicillin marker is introduced into the bacteria. pCP20 is an FLP recombinase that mediates the recombination between the FRT sites flanking the kanamycin cassette. The recombinase is temperature-sensitive and must be grown at temperatures below 30oC to introduce the plasmid into the cell. To complete the knockout the recombinase is cured at 42℃, removing it from the bacteria.[21] By performing a colony PCR over the created knockout mutants, the length should indicate a smaller fragment size than the WT ER2566.
SOPs used: SOP20
To create De novo synthesis system cell factories of psilocybin or cell factories producing it from 4-Hydroxyindol, triple transformants were created. The triple transformants were created in a stepwise process through a double transformant. The double ER2566 transformants were confirmed by negative selection on antibiotic LA-plates, whereas the triple transformants were confirmed using colony PCR with gene specific primers. If all there plasmids are correctly present in the cell, all three primer stets are able to bind in their respective PCR reaction. This results in the formation of three PCR products which the SnapGene predicted lengths.
Future Experiments
We also considered adding signal peptides to the sequence of both enzymes. Thereby PsiH and CPR would be transported to the cell membrane rather than floating around in the cytosol. Potentially, one might be able to preserve the native form in that way and thus greater activity, because P450 monooxygenases are often still partially misfolded by N-terminal modification - either as a result of the new N-terminal or simply because the protein is used to be closely associated with a membrane which is shown in the indelible hydrophobic domains.[12] However, this was an idea that came up late in the project and as we at some point plan to move the entire system to Rhodovulum sulfidophilum, which probably would require another signal peptide to work compared to E. coli, we never actually tested this, but it might be interesting in the future. In addition to this, it would also be interesting to investigate whether the use of a peptide linker between PsiH and CPR would optimize the production of psilocybin in terms of N-terminal modification. Hypothetically, this might have an impact by simply placing the two enzymes close to each other thereby making the transfer of electrons from CPR to the reaction catalyzed by PsiH more probable compared with the alternative scenario in which N-terminal modification that makes the enzymes more soluble also creates distance in between making the interaction more stochastic. Furthermore, one could improve tryptophan production in E. coli thereby obtaining a higher yield in psilocybin without adding tryptophan to the medium from start.[22]
[1] Fricke J, Blei F, Hoffmeister D. Enzymatic Synthesis of Psilocybin. Angewandte Chemie In-ternational Edition [Internet]. 2017 [cited 22 October 2021];56(40):12352-12355. Available from: https://pubmed.ncbi.nlm.nih.gov/28763571/
[2] Adams A, Kaplan N, Wei Z, Brinton J, Monnier C, Enacopol A et al. In vivo production of psilocybin in E. coli. Metabolic Engineering [Internet]. 2019 [cited 22 October 2021];56:111-119. Available from: https://pubmed.ncbi.nlm.nih.gov/31550507/
[3] Team:Sydney Australia - 2019.igem.org [Internet]. 2019.igem.org. 2021 [cited 22 October 2021]. Available from: https://2019.igem.org/Team:Sydney_Australia
[4] Hoefgen S, Lin J, Fricke J, Stroe M, Mattern D, Kufs J et al. Facile assembly and fluores-cence-based screening method for heterologous expression of biosynthetic pathways in fun-gi. Metabolic Engineering [Internet]. 2018 [cited 22 October 2021];48:44-51. Available from: https://pubmed.ncbi.nlm.nih.gov/29842926/
[5] Milne N, Thomsen P, Mølgaard Knudsen N, Rubaszka P, Kristensen M, Borodina I. Metabol-ic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and re-lated tryptamine derivatives. Metabolic Engineering [Internet]. 2020 [cited 22 October 2021];60:25-36. Available from: https://pubmed.ncbi.nlm.nih.gov/32224264/
[6] Hausjell J, Halbwirth H, Spadiut O. Recombinant production of eukaryotic cytochrome P450s in microbial cell factories. Bioscience Reports [Internet]. 2018 [cited 22 October 2021];38(2). Available from: https://pubmed.ncbi.nlm.nih.gov/29436484/
[7] Ichinose H, Wariishi H. High-level heterologous expression of fungal cytochrome P450s in Escherichia coli. Biochemical and Biophysical Research Communications [Internet]. 2013 [cit-ed 22 October 2021];438(2):289-294. Available from: https://pubmed.ncbi.nlm.nih.gov/23886957/
[8] Baek M, DiMaio F, Anishchenko I, Dauparas J, Ovchinnikov S, Lee G et al. Accurate prediction of protein structures and interactions using a three-track neural network. Science [Internet]. 2021 [cited 22 October 2021];373(6557):871-876. Available from: https://pubmed.ncbi.nlm.nih.gov/34282049/
[9] Mbaye M, Hou Q, Basu S, Teheux F, Pucci F, Rooman M. A comprehensive computational study of amino acid interactions in membrane proteins. Scientific Reports [Internet]. 2019;9(1). Available from: https://www.nature.com/articles/s41598-019-48541-2
[10] Hausjell J, Halbwirth H, Spadiut O. Recombinant production of eukaryotic cytochrome P450s in microbial cell factories. Bioscience Reports [Internet]. 2018 [cited 22 October 2021];38(2). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835717/
[11] Feyereisen R. Insect CYP Genes and P450 Enzymes. Reference Module in Life Sciences [Internet]. 2019 [cited 22 October 2021];. Available from: https://www.sciencedirect.com/science/article/pii/B9780128096338040401
[12] Ichinose H, Hatakeyama M, Yamauchi Y. Sequence modifications and heterologous ex-pression of eukaryotic cytochromes P450 in Escherichia coli. Journal of Bioscience and Bio-engineering [Internet]. 2015 [cited 22 October 2021];120(3):268-274. Available from: https://pubmed.ncbi.nlm.nih.gov/25732206/
[13] Kallunki, Barisic, Jäättelä, Liu. How to Choose the Right Inducible Gene Expression Sys-tem for Mammalian Studies?. Cells [Internet]. 2019 [cited 22 October 2021];8(8):796. Availa-ble from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721553/
[14] Brautaset T, Lale R, Valla S. Positively regulated bacterial expression systems. Microbial Biotechnology [Internet]. 2008 [cited 22 October 2021];2(1):15-30. Available from: https://pubmed.ncbi.nlm.nih.gov/21261879/
[15] Salcedo-Sora J, Jindal S, O'Hagan S, Kell D. A palette of fluorophores that are differentially accumulated by wild-type and mutant strains of Escherichia coli: surrogate ligands for profil-ing bacterial membrane transporters. Microbiology [Internet]. 2021 [cited 22 October 2021];167(2). Available from: https://pubmed.ncbi.nlm.nih.gov/33406033/
[16] Kell D, Yang L, Malla S, inventors. Engineered cells for production of indole-derivatives. WO2020187739. 2020 Sep 24.
[17] Bhandol H, Alindogan J, de Guzman A, Lim R. Review: Structure and regulation of the Acr efflux pumps and their role in antibiotic resistance in Escherichia coli [Internet]. UJEMI. 2021 [cited 22 October 2021]. Available from: https://ojs.library.ubc.ca/index.php/UJEMI/article/view/193259
[18] Hirakawa H, Inazumi Y, Masaki T, Hirata T, Yamaguchi A. Indole induces the expression of multidrug exporter genes in Escherichia coli. Molecular Microbiology [Internet]. 2004 [cited 22 October 2021];55(4):1113-1126. Available from: https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2004.04449.x
[19] Kneen M, Farinas J, Li Y, Verkman A. Green Fluorescent Protein as a Noninvasive Intracel-lular pH Indicator. Biophysical Journal [Internet]. 1998 [cited 22 October 2021];74(3):1591-1599. Available from: https://pubmed.ncbi.nlm.nih.gov/9512054/
[20] Janczak M, Bukowski M, Górecki A, Dubin G, Dubin A, Wladyka B. A systematic investiga-tion of the stability of green fluorescent protein fusion proteins. Acta Biochimica Polonica [Internet]. 2015 [cited 22 October 2021];62(3):407-411. Available from: https://pubmed.ncbi.nlm.nih.gov/26192770/
[21] Saragliadis A, Trunk T, Leo J. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. Journal of Visualized Experiments [Internet]. 2018 [cited 22 October 2021];(139). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235078/
[22] Niu H, Li R, Liang Q, Qi Q, Li Q, Gu P. Metabolic engineering for improving l-tryptophan production in Escherichia coli. Journal of Industrial Microbiology and Biotechnology [Inter-net]. 2019 [cited 22 October 2021];46(1):55-65. Available from: https://academic.oup.com/jimb/article/46/1/55/5996789?fbclid=IwAR2abxJn_iTnnqpNv6of1kJKSrZhZVi3slzhLAb0k2RH8ZRGqzcveoa-85o
Future Experiments
We also considered adding signal peptides to the sequence of both enzymes. Thereby PsiH and CPR would be transported to the cell membrane rather than floating around in the cytosol. Potentially, one might be able to preserve the native form in that way and thus greater activity, because P450 monooxygenases are often still partially misfolded by N-terminal modification - either as a result of the new N-terminal or simply because the protein is used to be closely associated with a membrane which is shown in the indelible hydrophobic domains.[12] However, this was an idea that came up late in the project and as we at some point plan to move the entire system to Rhodovulum sulfidophilum, which probably would require another signal peptide to work compared to E. coli, we never actually tested this, but it might be interesting in the future. In addition to this, it would also be interesting to investigate whether the use of a peptide linker between PsiH and CPR would optimize the production of psilocybin in terms of N-terminal modification. Hypothetically, this might have an impact by simply placing the two enzymes close to each other thereby making the transfer of electrons from CPR to the reaction catalyzed by PsiH more probable compared with the alternative scenario in which N-terminal modification that makes the enzymes more soluble also creates distance in between making the interaction more stochastic. Furthermore, one could improve tryptophan production in E. coli thereby obtaining a higher yield in psilocybin without adding tryptophan to the medium from start.[22]
References
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[2] Adams A, Kaplan N, Wei Z, Brinton J, Monnier C, Enacopol A et al. In vivo production of psilocybin in E. coli. Metabolic Engineering [Internet]. 2019 [cited 22 October 2021];56:111-119. Available from: https://pubmed.ncbi.nlm.nih.gov/31550507/
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[5] Milne N, Thomsen P, Mølgaard Knudsen N, Rubaszka P, Kristensen M, Borodina I. Metabol-ic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and re-lated tryptamine derivatives. Metabolic Engineering [Internet]. 2020 [cited 22 October 2021];60:25-36. Available from: https://pubmed.ncbi.nlm.nih.gov/32224264/
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[10] Hausjell J, Halbwirth H, Spadiut O. Recombinant production of eukaryotic cytochrome P450s in microbial cell factories. Bioscience Reports [Internet]. 2018 [cited 22 October 2021];38(2). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835717/
[11] Feyereisen R. Insect CYP Genes and P450 Enzymes. Reference Module in Life Sciences [Internet]. 2019 [cited 22 October 2021];. Available from: https://www.sciencedirect.com/science/article/pii/B9780128096338040401
[12] Ichinose H, Hatakeyama M, Yamauchi Y. Sequence modifications and heterologous ex-pression of eukaryotic cytochromes P450 in Escherichia coli. Journal of Bioscience and Bio-engineering [Internet]. 2015 [cited 22 October 2021];120(3):268-274. Available from: https://pubmed.ncbi.nlm.nih.gov/25732206/
[13] Kallunki, Barisic, Jäättelä, Liu. How to Choose the Right Inducible Gene Expression Sys-tem for Mammalian Studies?. Cells [Internet]. 2019 [cited 22 October 2021];8(8):796. Availa-ble from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721553/
[14] Brautaset T, Lale R, Valla S. Positively regulated bacterial expression systems. Microbial Biotechnology [Internet]. 2008 [cited 22 October 2021];2(1):15-30. Available from: https://pubmed.ncbi.nlm.nih.gov/21261879/
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[16] Kell D, Yang L, Malla S, inventors. Engineered cells for production of indole-derivatives. WO2020187739. 2020 Sep 24.
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[19] Kneen M, Farinas J, Li Y, Verkman A. Green Fluorescent Protein as a Noninvasive Intracel-lular pH Indicator. Biophysical Journal [Internet]. 1998 [cited 22 October 2021];74(3):1591-1599. Available from: https://pubmed.ncbi.nlm.nih.gov/9512054/
[20] Janczak M, Bukowski M, Górecki A, Dubin G, Dubin A, Wladyka B. A systematic investiga-tion of the stability of green fluorescent protein fusion proteins. Acta Biochimica Polonica [Internet]. 2015 [cited 22 October 2021];62(3):407-411. Available from: https://pubmed.ncbi.nlm.nih.gov/26192770/
[21] Saragliadis A, Trunk T, Leo J. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. Journal of Visualized Experiments [Internet]. 2018 [cited 22 October 2021];(139). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235078/
[22] Niu H, Li R, Liang Q, Qi Q, Li Q, Gu P. Metabolic engineering for improving l-tryptophan production in Escherichia coli. Journal of Industrial Microbiology and Biotechnology [Inter-net]. 2019 [cited 22 October 2021];46(1):55-65. Available from: https://academic.oup.com/jimb/article/46/1/55/5996789?fbclid=IwAR2abxJn_iTnnqpNv6of1kJKSrZhZVi3slzhLAb0k2RH8ZRGqzcveoa-85o
Results
