Part:BBa_K4365008

Signal peptide of HYPI from Aspergillus fumigatus

HYPI is a protein found in Aspergillus fumigatus and belongs to a family of highly secreted proteins called hydrophobins. The signal peptide from the protein led to no turboRFP secretion in Saccharomyces cerevisiae.

Collection of fungal signal peptides

	ID	Source	Species	Prediction coefficient	turboRFP secretion
sp1	BBa_K4365006	MPGI	Magnaporthe grisea	0.9562	Yes
sp2	BBa_K4365007	RodA	Aspergillus nidulans	0.9553	Yes
sp3	BBa_K4365008	HYPI	Aspergillus fumigatus	0.9703	No
sp4	BBa_K4365009	SsgA	Metarhizium anisopliae	0.9126	No
sp5	BBa_K4365010	HCF-4	Cladosporium fulvum	0.9126	Yes
sp6	BBa_K4365025	HCF-5	Cladosporium fulvum	0.9234	NA
sp7	BBa_K4365026	CCG-2	Neurospora crassa	0.9566	NA
sp8	BBa_K4365011	HCF-1	Cladosporium fulvum	0.6850	Yes
sp9	BBa_K4365027	HCF-2	Cladosporium fulvum	0.8144	NA
sp10	BBa_K4365012	HCF-3	Cladosporium fulvum	0.7934	No
sp11	BBa_K4365013	SC3	Schizophyllum commune	0.6327	No
sp12	BBa_K4365014	ABH3	Agaricus bisporus	0.9458	Yes
sp13	BBa_K4365028	COH1	Coprinus cinereus	0.8545	NA
sp14	BBa_K4365015	FBH1	Pleurotus ostreatus	0.3367	Yes
sp15	BBa_K4365016	Aa-PRI2	Agrocybe aegerita	0.6356	Yes
sp16	BBa_K4365033	Hyd-Pt1	Pisolithus tinctorius	0.8352	NA
sp17	BBa_K4365017	HFBI	Trichoderma reesei	0.9851	Yes
sp18	BBa_K4365018	HFBII	Trichoderma reesei	0.6862	Yes
sp19	BBa_K4365019	α-mating factor	Saccharomyces cerevisiae	0.9988	Yes

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Secretion signal peptides can be fused to a protein to direct it through the secretory pathway out of the cell . Such secretion of recombinant proteins facilitates the protein purification process and the removal of GMOs . However, commonly used signal sequences like the S. cerevisiae alpha-factor prepro-peptide K4365019 and the HSP150, are very long which makes their use in genetic constructs limited. K4365008 as a part of the collection of signal peptides represents a shorter sequence that could be inserted into the gene of interest simply by PCR and could display higher secretion efficiency.

The sequences of the hydrophobic signal peptide was collected from literature and was extracted via analysis of their sequence using the SignalP - 5.0 signal peptide predictor tool .

Identified sequence was codon-optimized for S. cerevisiae using the codon optimization tool on GenScript. Then it was synthesized by IDT as a gBlock together with a flexible glycine linker (K4365005) and the turboRFP gene (K4365020) .

Hydrophobins

Hydrophobins are a group of small (~100 amino acids) proteins that are expressed in ascomycetes and basidiomycetes, which are very efficiently secreted .

After secretion from the fungus, they can assemble on the cell walls to form a hydrophobic coating. For this reason, the functions of hydrophobins are related to cell surface activity. For example, they can cover hyphae so the surface tension can be lowered and the hyphae can breach the water/air interface, thus escaping from the aqueous environment and growing into the aerial hyphae . They also mediate the adhesion of the hyphae to hydrophobic surfaces such as in the case of pathogen-host interaction or symbiosis, as observed in lichens and in ectomycorrhizae (the symbiotic relationship between a fungal symbiont and the roots of a plant) . Hydrophobins also form a hydrophobic sheath to protect the surface of conidia, spores, and caps of fungi against wetting and desiccation .

Signal peptide secretion screening

Signal peptide siquence was cloned together with turboRFP K4365020 into the level 1 shuttle plasmid K4365022 (Figure 1).

Figure 1: fusion protein used for the secretion assays composed of a signal peptide, a flexible linker, and the turboRFP fluorescent protein. Created with Biorender.com.

After the transformation with the shuttle plasmid, the yeast colonies were grown on W0 medium plates supplemented with histidine, leucine, and methionine and were used for secretion assays in flasks and in a 48-well plate. The α-mating factor pre-pro signal peptide K4365019 was used as a positive control as it has been shown to be able to secrete proteins . A turboRFP without signal peptide was employed as a negative control to account for the amount of free turboRFP present in the media due to cell lysis.

Secretion measurement protocols:

• flask protocol
• 48-well plate protocol

The first secretion experiment was conducted in flasks and showed (Figure 2A) that cell lysis was low as indicated by the low fluorescence of the negative control. In order to have consistent results and ensure confidence we set up biological triplicates. For this, turboRFP secretion was measured using 48-well plate approach. Cells with induced expression were pipetted into the wells of the plate and grown overnight. Starting at 1 hour after induction, samples were taken and the growth rate was monitored by measuring turbidity (NTU) and the development of fluorescence in the supernatant was measured on a plate reader.

Despite the fact that K4365008 exhibited increased turboRFP secretion when measured using flasks a more sensitive approach showed that signal from K4365008 was on the level of cell lysis (Figure 2). Thus, this indicates that combination of K4365008 with turboRFP is not optimal for turboRFP secretion in S. cerevisiae.

Figure 2: A secretion by signal peptides in flasks. B secretion assay in 48-well plate with an initial cell density of 1 OD. The canonical α-mating factor pre-pro signal peptide (dark grey + control) was used as a positive control as it has been shown to be able to secrete proteins. A turboRFP without signal peptide was employed as a negative control to account for cell lysis (light grey, - control). The signal corresponding to K4365008 is depicted as a dashed line. Error bars represent mean ± SD.