For the future one could think of much more radical strategies for peroxisomal engineering with a final goal of a “minimal peroxisome” by redirecting metabolic pathways through changing the protein-localization-signal in the yeast genome. Additionally, endogenous metabolic pathways could be redirected to our novel artificial compartment for establishing a customized metabolism specifically tailored for the user's application.
Introduction
In order to achieve a fully controllable artificial compartment, the first step was to design a completely orthogonal import system. Next was the knockout of endogenous import systems. However, a few proteins are imported neither by the Pex5 nor the Pex7 import machinery. Therefore, specific genome engineering designs, such as knockouts, deleting or redirecting the protein localization could be utilized for the ultimate goal of creating a synthetic organelle.
Additionally, knockouts or genome integrations enable customization of the peroxisomal properties, such as membrane permeability, size/number, decoupling of peroxisomes from cytoskeleton and the peroxisomal metabolism.
All these strategies allow a rational design of an artificial compartment, which is fully engineerable regarding the proteome, metabolome and the entire peroxisomal environment.
Design of yeast multi -knockout strains
The Crispr Cas9 System
The demands on yeast engineering have significantly increased with the design of more complex systems and extensive metabolic pathways. Genetic techniques that have historically relied on marker recycling are unable to keep up with the ambitions of synthetic biologists. In recent years the Crispr Cas9 system has been used for several strain-engineering purposes, including:
- Markerless integration of multiple genetic cassettes into selected genomic loci
- Multiplexed and iterative gene knockouts without the need to recycle a marker
- Precise genome editing – nucleotide substitutions, etc.
We utilized the Cas9 system as a tool for peroxisomal engineering and have adopted the existing toolbox from (Lee et al. 2015) and the complete cloning system which also provides the possibilities for genome integration and gene editing by Cas9. For this, two oligonucleotides have to be designed for targeting the Cas protein to the gene of interest.
Two oligos, containing the targeting sequence of the gRNA, have to be annealed and can then be integrated in the gRNA entry Vector by a Golden Gate reaction. Adapted from (Lee et al. 2015)
Several gRNA vectors can subsequently be assembled into one vector with a Cas9 expression cassette and then be transformed into yeast. The expression of Cas9 together with gene specific gRNA´s leads to double strand breakage followed by non-homologous end joining repair or homologous recombination, in case of added repair DNA (figure 3).
Vector for Cas9 and gRNA expression, assembled by a Golden Gate reaction, containing a URA marker, Cen6 yeast origin and a kanamycin resistance. Adapted from (Lee et al. 2015)
The combination of the Cas9 system with DNA repair sequences enables not only knockouts of peroxisomal proteins, but also allows redirecting protein localization by changing protein targeting signals or integration of linear DNA into yeast chromosomes. Genome engineering facilitates yeast strain development for customized peroxisomes.
Repair DNA sequences can be used to increase the efficiency for Cas9 guided knocking out of specific genes, but would also allow genomic integration of targeting signals or complete genes. Adapted from (Lee et al. 2015)
The peroxisomal proteome of Saccharomyces cerevisiae
The peroxisomal proteome is studied extensively for saccharomyces cerevisiae and contains exactly 67 proteins (Kohlwein et al. 2013). The function is characterized for the most of those proteins and it is known, that yeast peroxisomes are expendable under optimal growth conditions. Nevertheless, some knockouts are lethal under oleate or stress conditions.
| Gene | Required for growth on oleate | Expression induced by oleate | Enzyme/activity | Molecular mass (kDa) | Isoelectric point | Molecules per cell | Localization | Function |
|---|---|---|---|---|---|---|---|---|
| ß-Oxidation enzymes | ||||||||
| PCS60 (FAT2) | No | Yes | Medium chain fatty acyl-CoA synthetase | 60.5 | 9.98 | 8.770 | Peripheral peroxisomal membrane and matrix | Activates fatty acids with a preference for medium chain lengths, C9-C13 |
| FAT1 | No | - | Very long chain fatty acyl-CoA synthetase and long chain fatty acid transporter | 77.1 | 8.47 | 16,900 | Lipid droplet, ER, peroxisome Three predicted TM | Activates fatty acids with a preference for very long chain lengths, C20–C26 |
| POX1 | Yes | Yes | Acyl-CoA- oxidase | 84.0 | 8.73 | ND | Peroxisomal matrix | Oxidation of acyl-CoA |
| CTA1 | No | Yes | Catalase | 58.6 | 7.46 | 623 | Peroxisomal matrix | Degrades hydrogen peroxide produced by Pox1 |
| FOX2 (POX2) | Yes | Yes | Multifunctional enzyme; 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase | 98.7 | 9.75 | ND | Peroxisomal matrix | - |
| POT1 (FOX3, POX3) | Yes | Yes | 3-Ketoacyl-CoA thiolase | 44.7 | 7.56 | ND | Peroxisomal matrix | Cleaves 3-ketoacyl-CoA into acyl-CoA and acetyl-CoA |
| DCI1 (ECI2) | - | - | Δ(3,5)-Δ(2,4)-dienoyl-CoA isomerase (putative) | 30.1 | 8.83 | ND | Peroxisomal matrix | Auxiliary enzyme of fatty acid β-oxidation; role in β-oxidation debated |
| SPS19 (SPX1) | Yes | Yes | 2,4-Dienoyl-CoA reductase | 31.1 | 9.67 | ND | Peroxisomal matrix | Auxiliary enzyme of fatty acid β-oxidation |
| ECI1 | Yes | Yes | Δ3, Δ2-enoyl-CoA isomerase | 31.7 | 8.21 | ND | Peroxisomal matrix | Auxiliary enzyme of fatty acid β-oxidation |
| TES1 (PTE1) | Yes | Yes | Acyl-CoA thioesterase | 40.3 | 9.58 | ND | Peroxisomal matrix | Auxiliary enzyme of fatty acid β-oxidation |
| MDH3 | Yes | Yes | Malate dehydrogenase | 37.3 | 10.00 | 3,300 | Peroxisomal matrix | Required for the malate-oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+, part of the glyoxylate cycle |
| IDP3 | Yes | Yes | NADP+ dependent isocitrate dehydrogenase | 47.91 | 10.02 | ND | Peroxisomal matrix | Required for the 2-ketoglutarate/isocitrate shuttle, exchanging peroxisomal NADP+ for cytosolic NADPH |
| CAT2 | No | No | Carnitine acetyl-CoA transferase | 77.2 | 8.34 | 470 | Peroxisome mitochondria | Transfers activated acetyl groups to carnitine to form acetylcarnitine which can be shuttled across membranes |
| Glyoxylate cycle | ||||||||
| CIT2 | No | - | Citrate synthase | 51.4 | 6.34 | 2,310 | Peroxisomal matrix | Condensation of acetyl CoA and oxaloacetate to form citrate |
| MDH3 | Yes | Yes | Malate dehydrogenase | 37.3 | 10.00 | 3,300 | Peroxisomal matrix | Required for the malate–oxaloacetete shuttle, to exchange peroxisomal NADH for cytosolic NAD+ |
| MLS1 | Yes | - | Malate synthase | 62.8 | 7.18 | ND | Peroxisomal protein | Required for utilization of nonfermentable carbon sources |
| Other peroxisome-associated enzyme activities | ||||||||
| GPD1 (DAR1, HOR1, OSG1, OSR5 | - | - | NAD-dependent glycerol-3-phosphate dehydrogenase | 42.9 | 5.26 | 807 | Peroxisome, cytosol, nucleus | Key enzyme of glycerol synthesis, essential for growth under osmotic stress |
| PNC1 | - | - | Nicotinamidase | 25.0 | 6.23 | 7,720 | Peroxisome, cytosol | Converts nicotinamide to nicotinic acid as part of the NAD(+) salvage pathway |
| NPY1 | - | - | NADH diphosphatase | 43.5 | 6.26 | 846 | Peroxisome cytosol | Hydrolyzes the pyrophosphate linkage in NADH and related nucleotides |
| STR3 | - | - | Cystathionine β-lyase | 51.8 | 7.96 | ND | Peroxisome | Converts cystathionine into homocysteine |
| STR3 | - | - | Cystathionine ß-lyase | 51.8 | 7.96 | ND | Peroxisome | Converts cystathionine into homocysteine |
| GTO1 | - | - | ω-Class glutathione transferase | 41.3 | 9.53 | - | Peroxisome | Induced under oxidative stress |
| AAT2(ASP5) | - | Yes | Aspartate aminotransferase | 46.1 | 8.50 | 7,700 | Cytosol, peroxisome | Involved in nitrogen metabolism |
| PCD1 | - | - | Nudix pyrophosphatase with specificity for coenzyme A and CoA derivatives | 39.8 | 6.59 | 238 | Peroxisome | May function to remove potentially toxic oxidized CoA disulfide from peroxisomes |
| LPX1 | - | Yes | Triacylglycerol lipase | 43.7 | 8.16 | 2,350 | Peroxisomal matrix | - |
| Peroxisomal transporters | ||||||||
| PXA1 (LPI1, PAL1, PAT2, SSH2 | - | - | Subunit of a heterodimeric ATP-binding cassette transporter complex | 100.0 | 10.34 | ND | Peroxisomal membrane | Import of long-chain fatty acids into peroxisomes |
| PXA2 (PAT1) | - | - | Subunit of a heterodimeric ATP-binding cassette transporter complex | 97.1 | 9.47 | ND | Peroxisomal membrane | Import of long-chain fatty acids into peroxisomes |
| ANT1(YPR118C) | - | - | Adenine nucleotide transporter | 36.4 | 10.6 | 2,250 | Peroxisomal membrane | Involved in β-oxidation of medium-chain fatty acids |
| Peroxins | ||||||||
| Pex1 (PAS1) | - | - | AAA ATPase | 117.3 | 6.93 | 2,100 | Peroxisomal membrane | Involved in recycling of Pex5, forms heterodimer with Pex6 |
| Pex2 (RT1, PAS5) | - | - | E3 ubiquitin ligase | 30.8 | 9.02 | 339 | Peroxisomal membrane | RING finger protein, forms complex with Pex10 and Pex12. Involved in matrix protein import |
| Pex3 (PAS3) | - | - | - | 50.7 | 6.29 | 1,400 | Peroxisomal membrane | Required for proper localization of PMPs |
| Pex4 (PAS2, UBC10) | - | - | Ubiquitin conjugating enzyme | 21.1 | 5.36 | ND | Peroxisomal membrane | Involved in matrix protein import |
| Pex5 (PAS10) | - | - | Soluble PTS1 receptor | 69.3 | 4.79 | 2,070 | Cytosol and peroxisomal matrix | Required for import of PTS1-containing peroxisomal proteins, contains TPR domains |
| Pex6 (PAS8) | - | - | AAA ATPase | 115.6 | 5.44 | 1,630 | Peroxisomal membrane | Involved in recycling of Pex5, forms heterodimer with Pex1 |
| Pex7 (PAS7, PEB1) | - | - | Soluble PTS2 receptor | 42.3 | 8.34 | 589 | Cytosol and peroxisomal matrix | Requires Pex18 and Pex21 for association to the receptor docking site, contains WD40 repeat |
| Pex8 (PAS6) | - | - | Intra peroxisomal organizer of the peroxisomal import machinery | 68.2 | 7.62 | 538 | Peroxisomal matrix and luminal membrane face | Pex5-cargo dissociation |
| Pex9 | - | - | PTS-receptor | - | - | - | - | - |
| Pex10 | - | - | E3 ubiquitin ligase | 39.1 | 9.88 | ND | Peroxisomal membrane | RING finger protein involved in Ubc4-dependent Pex5 ubiquitination. Forms complex with Pex2 and Pex12 |
| Pex11 (PMP24, PMP 27) | - | - | - | 26.9 | 10.65 | 1,630 | Peroxisomal membrane | Involved in peroxisome fission, required for medium-chain fatty acid oxidation |
| Pex12 (PAS11) | - | - | E3 ubiquitin ligase | 46.0 | 9.86 | 907 | - | RING finger protein, forms complex with Pex2 and Pex10 |
| Pex13 (PAS20) | - | - | Component of docking complex for Pex5 and Pex7 | 42.7 | 9.83 | 7,900 | Peroxisomal membrane | Forms complex with Pex14 and Pex17 |
| Pex14 | - | - | Central component of the receptor docking complex | 38.4 | 4.61 | 2,570 | Peroxisomal membrane | Interacts with Pex13 |
| Pex15 (PAS21) | - | - | - | 43.7 | 8.42 | 1,070 | Peroxisomal membrane | Recruitment of Pex6 to the peroxisomal membrane, tail anchored PMP |
| Pex17 (PAS9) | - | - | Component of docking complex for Pex5 and Pex7 | 23.2 | 10.24 | 656 | Peroxisomal membrane | Forms complex with Pex13 and Pex14 |
| Pex18 | - | - | - | Required for PTS2 import | 32.0 | 4.78 | ND | Interacts with Pex7 partially redundant with Pex21 |
| Pex19 (PAS12) | - | - | Chaperone and import receptor for newly synthesized PMP | 38.7 | 4.08 | 5,350 | Cytosol, peroxisome, farnesylated | Interacts with PMPs, involved in PMP sorting. Also interacts with Myo2 and contributes to peroxisome partitioning |
| Pex21 | - | - | Required for PTS2 protein import | 33.0 | 6.67 | ND | Cytosol | Interacts with Pex7, partially redundant with Pex18 |
| Pex22(YAF5) | - | - | Required for import of peroxisomal proteins | 19.9 | 8.33 | 259 | Peroxisomal membrane | Recruits Pex4 to the peroxisomal membrane |
| Pex25 | - | - | Involved in the regulation of peroxisome size and maintenance, required for re-introduction of peroxisomes in peroxisome deficient cells | 44.9 | 9.77 | 2,420 | Peripheral peroxisomal membrane | Recruits GTPase RhoI to peroxisomes, interacts with homologous protein Pex27 |
| Pex27 | - | - | Involved in the regulation of peroxisome size and number | 44.1 | 10.49 | 382 | Peripheral peroxisomal membrane | Interacts with homologous protein Pex25 |
| Pex28 | - | - | Involved in the regulation of peroxisome size, number and distribution | 66.1 | 7.09 | ND | Peroxisomal membrane | May act upstream of Pex30, Pex31 and Pex 32 |
| Pex29 | - | - | 63.5 | 6.8 | 5,040 | Peroxisomal membrane | May act upstream of Pex30, Pex31 and Pex32 | |
| Pex30 | - | - | Involved in the regulation of peroxisome number | 59.5 | 5.59 | 4,570 | Peroxisomal membrane | Negative regulator, partially functionally redundant with Pex31 and Pex32 |
| Pex31 | - | - | Involved in the regulation of peroxisome number | 52.9 | 10.15 | 238 | Peroxisomal membrane | Negative regulator, partially functionally redundant with Pex30 and Pex32 |
| Pex32 | - | - | Involved in the regulation of peroxisome number | 48.6 | 9.14 | ND | Peroxisomal membrane | Negative regulator partially functionally redundant with Pex30 and Pex31 |
| Pex34 | - | - | Involved in the regulation of peroxisome number | 16.6 | 10.30 | ND | Peroxisomal membrane | - |
| Peroxisome fission and inheritance | ||||||||
| DYN2 (SLC1) | - | - | Light chain dynein | 10.4 | 9.03 | 1,310 | Cytosol | Microtubule motor protein |
| SEC20 | - | - | v-SNARE | 43.9 | 5.92 | 4,910 | Golgi, ER | Involved in retrograde transport from the Golgi to the ER, interacts with the Dsl1 complex through Tip20 |
| SEC39(DSL3) | - | - | Component of the Ds11p-tethering complex | 82.4 | 4.65 | 1,840 | ER, nuclear envelope | Proposed to be involved in protein secretion |
| DSL1 (RNS1) | - | - | Component of the ER target site that interacts with coatomer | 88.1 | 4.69 | 8,970 | Peripheral ER, Golgi membrane | Forms a complex with Sec39 and Tip20 that interacts with ER SNAREs, Sec20 and Use1 |
| FIS1 (MDV2) | - | - | Required for peroxisome fission | 17.7 | 9.87 | 2,410 | Peroxisomal membrane mitochondria | Tail anchored protein recruits Dnm1 via Mdv1/Caf4; also involved in mitochondrial fission |
| DNM1 | - | - | GTPase, dynamin like protein involved in peroxisome fission | 85.0 | 5.25 | 9,620 | - | Also involved in mitochondrial fission |
| VPS1 (GRD1, LAM1, SPO15, VPL1, VPT26) | - | - | GTPase, dynamin like protein involved in peroxisome fission | 78.7 | 8.15 | 5,960 | - | Also involved in vacuolar protein sorting |
| VPS34 (END12, PEP15, VPL7, VPT29, STT8, VPS7) | - | - | Phosphatidylinositol 3-kinase | 100.9 | 7.79 | 1,080 | - | Forms complex with Vps15 |
| INP1 | - | - | Involved in retention of peroxisomes in mother cells | 47.3 | 8.34 | 639 | Peroxisomal membrane | Recruited to the peroxisome by binding to Pex3 |
| INP2 | - | - | Myo2 receptor, involved in peroxisome inheritance | 81.5 | 9.41 | 736 | Peroxisomal membrane | - |
| RHO1 | - | - | GTP binding protein of the Rho subfamily of Ras like proteins, involved in actin assembly at the peroxisome | 23.2 | 6.07 | ND | - | Involved in de novo peroxisome formation recruited to peroxisomes by Pex25 |
Pex9 is a recently discovered import receptor for PTS1 proteins, which is induced by oleate and is an import receptor for both malate synthase isoenzymes Mls1p and Mls2p. In order to get a completely empty reaction room, a Pex9 knockout was designed to prevent unintended protein import.
Pex 31 & Pex 32
It has been shown that knockouts of Pex31 and Pex32 leads to an increased Peroxisomal size, but additionally the membrane permeability was affected (Zhou et al. 2016). This effect can be used as a tool for engineering membrane permeability by knocking out or overexpress both genes. A knockout would lead to an increased permeability and one could think of an opposite effect in case of overexpression, but this has not been shown yet.
INP1
The INP1 knockout was designed after the skype call of Prof Dueber, who recommended us, decoupling of peroxisomes from cytoskeleton in order to improve the secretion efficiency. INP1 is responsible for the tethering of peroxisome, which would inhibit the secretion of peroxisomes.
POT1
The only protein, which is imported by the Pex7 import machinery in saccharomyces is the 3-ketoacyl-CoA thiolase (POT1). A knockout of POT1 would enable utilizing the Pex7 import for proteins of interest, which cannot be tagged at the C-terminus with pts1, without having unintended import of other enzymes.
Genomic integration of our novel Pex5 import receptor
After testing our new Pex5 import systems, which is completely orthogonal to the natural import, the next step would be to replace the endogenous system with our artificial import system. Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance (Figure 4). Afterwards the plasmid was transformed into the yeast strain which was created by our collaboration partner Aachen (double knockout strain Pex5 & Pex7).
Therefore, an integration plasmid was designed with help of the previously described yeast toolbox, containing HO locus homologies and a hygromycin resistance
The resulting yeast strain allows full control over the peroxisomal matrix proteome, by replacing the whole protein import machinery, which is the first step for creating our artificial compartment.
Outlook
Besides the genome engineering approaches, which were performed in our project one could think of more radical strategies for peroxisomal engineering. A final goal could be a “minimal peroxisome”, which contains only the proteins that are required for the biogenesis of the peroxisome and import of proteins and metabolites. On the one hand peroxisomal pathways could be redirected to cytosol or other organelles and one the other hand endogenous metabolic pathways could be redirected to our novel artificial compartment by changing the protein localization signal in the yeast genome with help of the Cas9 system. All these strategies would allow tremendous improvements for metabolic engineering applications by creating an artificial compartment, which can be rational designed and customized for specific metabolic pathways.
Vector for Cas9 and gRNA expression, assembled by a Golden Gate reaction, containing a URA marker, Cen6 yeast origin and a kanamycin resistance. Adapted from (Lee et al. 2015)
The combination of the Cas9 system with DNA repair sequences enables not only knockouts of peroxisomal proteins, but also allows redirecting protein localization by changing protein targeting signals or integration of linear DNA into yeast chromosomes. Genome engineering facilitates yeast strain development for customized peroxisomes.
Repair DNA sequences can be used to increase the efficiency for Cas9 guided knocking out of specific genes, but would also allow genomic integration of targeting signals or complete genes. Adapted from (Lee et al. 2015)
The peroxisomal proteome of Saccharomyces cerevisiae
The peroxisomal proteome is studied extensively for saccharomyces cerevisiae and contains exactly 67 proteins (Kohlwein et al. 2013). The function is characterized for the most of those proteins and it is known, that yeast peroxisomes are expendable under optimal growth conditions. Nevertheless, some knockouts are lethal under oleate or stress conditions.
