Week 2 HW: DNA Design
Hydrophobin Overexpression in Pleurotus ostreatus
Part 0: Setup in Kernel
Repository: Rebeka_Palfi_week-2_project
Part 1: Expression target
In HW1 I described my concept about connecting the food and packaging production in a mushroom farm. In case of packaging solutions their behavior under wet and humid conditions are critical in many fields of the industry. So my goal in HW 2 is to overexpress the mushroom’s native hydrophobin. I chose Vmh2, a class I hydrophobin native to Pleurotus ostreatus, expressed specifically in vegetative (non-fruiting) mycelium (Peñas et al. 2002).
As overexpressig hydrophobin can affect the mechanical properties in nn advantegous ways, my plan is to apply this modified strain only to the second, post-harvest growth phase of the already-exhausted substrate block, after the edible fruiting flush is harvested and the block is intervowen by the non modified mycelium. I hope to build up a water-repellent surface layer. The edible fruiting fungus itself stays unmodified (non-GMO).
The host organism would be P. ostreatus itself — homologous overexpression, not a heterologous host.
As my knowledge in this field is not well established, I used Claude AI to collect relevant data, literature and to help me identify the best solutions to my problem. Regarding localization Claude recommended this: “The native N-terminal signal peptide is retained so the protein exits the cell and self-assembles extracellularly, as in the wild type.”
Part 2: Genetic parts
Collecting the parts I might need to achieve my goal is also done with the help of Claude AI.
| Part | Choice | Why this part is needed here (source) |
|---|---|---|
| Promoter | P. ostreatus native gpd promoter (with its native intron) | A promoter is required to initiate transcription of the downstream CDS. The native gpd promoter was shown to drive strong, constitutive expression of a fused reporter/marker gene in P. ostreatus itself (Ding et al. 2011; Fabriki-Ourang et al. 2013) |
| 5’UTR | Carried with the gpd promoter fragment (contains one intron) | Sits between the transcription start site and the start codon. In related mushroom species, removing the intron from this region abolished reporter expression — i.e. it is required, not merely optional (Burns et al. 2005) |
| CDS | vmh2, genomic sequence (with introns) | This is the sequence that actually encodes Vmh2; its exon/intron structure and coding boundaries were determined directly for this gene (Peñas et al. 2002) |
| Signal peptide | Native Vmh2 N-terminal signal peptide, encoded as part of the CDS | Needed so the protein enters the secretory pathway and reaches the cell surface. Peñas et al. (2002) identified a region compatible with a signal peptide directly within the Vmh2 sequence — the gene already encodes one; it is not an added part |
| Terminator | CaMV 35S | Needed to terminate transcription and add the poly-A signal. Already used successfully downstream of a gpd-driven cassette delivered by Agrobacterium into P. ostreatus (Fabriki-Ourang et al. 2013) |
| Selection marker | hph, own CaMV 35S promoter/terminator cassette | Needed to distinguish successfully transformed cells from the (majority) untransformed ones. The same marker/cassette design is already validated with Agrobacterium delivery into P. ostreatus (Ding et al. 2011; Fabriki-Ourang et al. 2013) |
| Backbone | pCAMBIA1300-type T-DNA binary vector | Needed to physically carry and deliver the cassette into the fungal genome via Agrobacterium. T-DNA-mediated delivery into P. ostreatus mycelium is directly demonstrated (Ding et al. 2011) |
Part 3: Assembled construct
Finding the right part in Kernel was difficult for me, I wasn’t able to finalize the construct as I planned in time.
Part 4: Optimize and quality-check
I suppose codon optimization won’t be necessery as I would work with a homologous host. I still need to build the construct properly to be able to quality check it.
Part 5: From design to build
Assembly: Gibson assembly (Gibson et al. 2009) to join the four fragments — promoter, CDS, terminator, marker cassette — into the backbone. Sequence-independent and isothermal, so it avoids searching for compatible restriction sites across a GC-rich, intron-containing native fragment.
Synthesis fidelity: de novo gene synthesis error rates increase with sequence length and complexity (Tian et al. 2004). Vmh2’s short, cysteine-rich, repetitive coding sequence warrants full sequencing verification of the synthesized fragment before assembly.
Prototype vs. scale: small-scale Agrobacterium-mediated transformation first (following the Ding et al. 2011 / Fabriki-Ourang et al. 2013 protocols), hygromycin selection, then PCR and Southern blot to confirm single-copy integration, before scaling the confirmed strain to a production-size inoculum for the second colonization phase.
Part 6: Open questions and risks
Overexpression assumption: Disruption studies (Han et al. 2023) show that losing Vmh2/Vmh3 reduces surface hydrophobicity, but whether overexpression proportionally increases it is untested.
Sequence verification: Still need direct confirmation in Kernel before the final build.
AI use disclosure
Claude (Anthropic) was used as a tutor and research assistant across multiple sessions to: (1) develop the construct concept from the HF1 project; (2) evaluate and reject a laccase-based alternative; (3) select Vmh2 and justify the homologous-overexpression strategy; (4) research and cite genetic parts (promoter, terminator, backbone, marker) against literature precedent; (5) reason through the assembly order and justify it.
Sources
- Peñas, M.M., Rust, B., Larraya, L.M., Ramírez, L., Pisabarro, A.G. (2002). Differentially Regulated, Vegetative-Mycelium-Specific Hydrophobins of the Edible Basidiomycete Pleurotus ostreatus. Appl. Environ. Microbiol. 68(8):3891-3898. Link
- Han, J., Kawauchi, M., Schiphof, K., Terauchi, Y., Yoshimi, A., Tanaka, C., Nakazawa, T., Honda, Y. (2023). Features of disruption mutants of genes encoding for hydrophobin Vmh2 and Vmh3 in mycelial formation and resistance to environmental stress in Pleurotus ostreatus. FEMS Microbiology Letters 370:fnad036. Link
- Ding, Y., Liang, S., Lei, J., Chen, L., Kothe, E., Ma, A. (2011). Agrobacterium tumefaciens mediated fused egfp-hph gene expression under the control of gpd promoter in Pleurotus ostreatus. Microbiol. Res. 166:314-322. DOI
- Fabriki-Ourang, S., Jalali-Javaran, M., Mohammadi-Goltapeh, E., Alizadeh, H., Honari, H. (2013). Optimization of Agrobacterium-mediated transformation in oyster mushroom (Pleurotus ostreatus) by vector containing human pro-insulin gene. Iranian Journal of Genetics and Plant Breeding 2(1). PDF
- Burns, C., Gregory, K.E., Kirby, M., Cheung, M.K., Riquelme, M., Elliott, T.J., Challen, M.P., Bailey, A., Foster, G.D. (2005). Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns. Fungal Genet. Biol. 42(3):191-199. DOI
- Gibson, D.G., Young, L., Chuang, R.-Y., Venter, J.C., Hutchison, C.A., Smith, H.O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6:343-345. DOI
- Tian, J., Gong, H., Sheng, N., Zhou, X., Gulari, E., Gao, X., Church, G. (2004). Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432:1050-1054. DOI