Hydrophobins Production

HYDROPHOBINS PRODUCTION: A BOTTLENECK TO COMMERCIAL USES

ABSTRACT

Hydrohobins are surface active proteins, they are produced by ascomycetes and Basidiomycetes, they help the fungi to escape from the aqueous environment, protecting the fungi against desiccation and helping the fungi to colonize the outer environment, hydrphpobins have amphiphilic nature, due to this characteristic, they have particular and interesting properties with several industrial applications, like drug availability and protein purification. However, the production of these proteins beyond lab scale has become a bottleneck for further industrial applications, homologous and heterologous methods have been used to produce hydrophobins, but the yield of the process is relatively low. This review makes an overview of the technologies and methods used to enhance the production of these interesting molecules.

1. Introduction2. Classification of hydrophobines

2.1. Class I Hydrophobins2.2. Class II Hydrophobins

3. Properties of hydrophobines3.1. Hydrophobins self assemble3.2. Hydrophobins multimerization3.3. Physical properties of hydrophobins 4. Production of hydrophobins4.1. Homologous methods4.1.1 Class I production4.1.2 Class II production4.2. Heterologous methods4.2.1. Prokaryotes expression 4.2.2. Eukaryotes expression4.2.2.1 Plants and insects production5. Extraction and purification6. conclusion

1. INTRODUCTION

Hydrohobins are very unique proteins they are amphiphilic molecules, they have an hydropholic part and a hydrophobic part. When they are secreted in the medium they self-assemble in membranes protecting the hydrophobic cell wall and making the environment hydrophobic (Wosten et al. 1997), the change of the nature of the aerial hyphae (from hydrophilic to hydrophobic) allows the fungi to colonize other environments, helping to grow their hyphaes and spread the spores by wind.

Hydrophobins are small proteins produced by filamentous fungi and dimorphic yeast (Basiodiomycetes, Ascomycetes and Zygomycetes)(Scholtmeijer et al., 2001). They are globular with a molecular weight between 7 and 15 kd.(Linder et al., 2005, Scholmeijer et al., 2001). Several genes have been identified from a great number of fungi species producing active hydrophobins, one of the first hydrophobin gene was discovered during the study of the development of Schizophyllum commune (Wessel et al. 1991) These proteins help in the growing and spreading of hyphae, spores and fruiting bodies (Wösten, 2001), hydrophobins are secreted by hyphae, then they form a stable monolayer on the surface and when they are in contact with hydrophilic/hydrophobic interfaces they confer hydrophobicity to fungal surfaces in contact with air (Mc Cabe & Alfen,2009). These hydrophobins improved attachment of hyphae to hydrophobic surfaces.(Wösten, 1999).

Some hydrophobins seemed to have involved in certain pathogenesis like Aspergillus fumigatus in lung diseases, hydrophobins cover the conidia surface giving resistance against alveolar macrophages (paris et al., 2003)(Abad et al., 2010), in plant pathogenesis hydrophobins are important in the spread of the disease, e.g. Cladosporium fulvum a phytophatogenic fungi which could express class I and class II hydrophobins it is the main agent in tomato leaf mold disease, the mechanism by this fungi attacks the plant is mediated for one hydrophobin (HCF1),helping to dispersion of the spores by water droplets (Spanu, 1997)(Segers et al., 1999)(Nielsen et al., 2001)

Hydrophobins comply an important role during the life cycle of the fungi, they decrease the water-surface tension of the medium, protecting the fungi against desiccation (making the surface of the hyphae, spores and conidia hydrophobic)(Shokribousjein et al., 2011) and permit unidirectional passage of nutrients from the hydrophobic side to hydrophilic (up to 10,000 Da) and prevent passage of molecules over 10,000 Da from the hydrophilic side (Linder et al., 2005), hydrophobines are able to self assemble at hydrophilic-hydrophobic interfaces into an amphiphatic membrane, resulting on a change of the nature of the target surface (Armenante, 2008).

These properties have raised the scientific interest due to the applications possibilities, however production of hydrophobins in high amounts remained difficult, here we discussed the methods, the microorganisms and the techniques used to produce these proteins.

2. Classification of hydrophobins

Hydrophobins are classified in two classes, class I and class II, this is based on its solubility and sequence comparison (Linder et al., 2005)(Sarlin et al., 2005). There are about 70 and increasing hydrophobins genes found in databases, a common characteristic is that all of these proteins share eight well conserved cysteine residues in the structure of the finished protein. Class I hydrophobins have more aminoacids (100-125) than Class II (50-100), which lead to different properties, and different conformations according the microorganism(Fig. 1), due to the presence of more β-sheets, in class I hydrophobins they tend to form fibers(polymers) or aggregates (rodlets). Class II have a globular shape consisting on an anti parallel β-barrel formed by two β-hairpins connected by a stretch α-helix, there are 4 disulfide bonds giving high stability to the structure (Fig.2).

Fig. 1 Comparison between the sequences of some class I and Class II hydrophobins, only the aminacids between the first and last Cys residues are shown due to the high sequence variation at the termini. The eight cysteine residues are highlighted in yellow whit the disulfide bonds position in brackets. (Armenante, Doctoral thesis, 2010)

2.1. Class I Hydrophobins

Weseels et al, (1991), coined the term hydrophobin, working over a gene coded by S. commune, the product of this gene was a protein with 125 aminoacids, which was insoluble in water and forms aggregates. Subsequent studies showed that this type of hydrophobins (class I) are soluble in the medium at the beginning, but in contact with air or agitation they form aggregates, only when they are dissolved in trifluoroacetic acid and it was evaporated they become soluble in water again (Linder et al, 2005) (Wösten, 2001). There are two types whitin Class I, Class Ia and class Ib the first is produced by Ascomycetes and the others are from Basidiomycetes, class Ib are more hydrophobic than class Ia, due to differences in their structure and assemble (Linder et al., 2005).

The basic structure of class I hydrophobin are formed by a four stranded β-barrel with and additional two stranded antiparallel β-barrel linked to one face, the complete frame has four disulfide bridges which gives strength to the structure, two of the disulfide bridges are located at the centre of the barrel and the other two connect the outside of the barrel to and additional sheet of a nearby loop (fig. 2), the amphipathic core structure and the additional Cys 3-Cys4 loop help to form additional β-structures leading to rodlet formation (Armenante. 2010) All class I hydrophobin share the central core structure with the eight cysteine residues, but there is a high degree of sequence variation across the family (kwan et al., 2008)

2.2. Class II Hydrophobins

Class II have a hydrophobic part which is made of 2 b-hairpins including only aliphatic amino acids and a hydrophilic part which includes one a-helix. Class I are similar to class II but without a-helix (kallio et al., 2007) and with more amino acids and diversity in amino acid sequences (Linder et al, 2005). The basic structure of class II hydrophobins consist in antiparallel β-barrel formed by two β-hairpins connected by a stretch of α-helix, class II hydrophobins have four disulfide bridges as class I, one is connecting the outside of the barrel with the α-helix, a second one act as a cross link between the two β-hairpins, these two disulfide bridges are completely enclosed in the barrel and they are in opposite ends giving high stability to the frame (Fig. 2), class II hydrophobins is a more uniform group than class I, so, the similarities between species could vary from 25-95% which makes easy to compare the properties with the structure of HFBI hydrophobin from T. reesei, which is the most well characterized hydrophobin.

Class II have lower tendency to form aggregates, they are soluble in water and in organic solvents e.g. Ethanol (60%)(kisko, 2008) or hot sodium dodecylsulfate (SDS 2%)(Scholtmeijer et al, 2001), they show more tendency towards foaming, production levels at laboratory scale are higher class II are higher than class I(Linder et al, 2005) but Class I adhere more to surfaces than class II(Linder, 2009)

Fig 2. Comparisson of the structures between HFBII hydrophobin from T. reesei(class II) on the left (Linder et al. 2005) right, EAS hydrophobin (Class I) from N. crassa (Kwan, et al, 2006)

3. Properties of Hydrophobins

3.1. Hydrophobins self assemble

One of the most important properties of hydrophobins is the tendency to aggregations on interfaces, these can occur between solid/liquid, Liquid/liquid and Liquid/vapour, hydrophobins can form various types of self-assembled structures. Regardless of the structure both classes have the ability to decrease the surface tension of surfaces either by stabilizing oil droplets in water by forming a film around the droplet (class I) or making emulsions in oil/water interfaces (class II).

The high capacity to decrease the surface tension of surfaces of hydrophobins is due to the amphiphilic nature of these proteins, amphiphilic proteins have the capacity to migrate to hydrophobic-hydrophilic interfaces (air/water) and to stabilize and dissolve hydrophobic molecules in water (linder et al., 2005). At laboratory scale hydrophobins form two types of multimers, in solution and aggregates (rodlets).

3.2. Hydrophobins multimerization

Class I hydrophobins tend to form rodlets In vitro the diameter of this structures is about 10 nm, this process start with a α-rich conformation at the water-air interface, subsequently the polymerization continues and the hydrophobin layer pass to a β-rich rodlet conformation, once the hydrophobin is attached to a surface the binding is very resistant to be detached. Class II hydrophobins do not have rodlet conformation, they form a stable, planar and organized film near to a crystalline form, this hydrophobins in solution forms multimers which act as a detergent micelles with a amphiphilic nature (Armenante, 2010)(Linder et al., 2005)

When a Class I and II hydrophobins is purified its tends to form rodlets and aggregates, and become water insoluble, however when some cyclic carbohydrates (α,β,γ Cyclodectrins and its homologues) interact with the protein and affect the water solubility increasing its solubility, this phenomena have been described in SC3 hydrophobin from S. commune where its N-terminal region is linked to a residue of 20 mannose residues making this part more hydrophilic (Armenante et al., 2010), hydrophobins suffered post translational modifications creating disulfide bridges in the structure between the cysteine residues creating there four loops, thus forming a stable and durable structure to pass through the secretory pathway to the medium (Whiteford & Spanu, 2002).

Fig 3, schematic representation of the multimerization of hydrophobins and the formation of surface films on water/air interface (Kallio et al., 2007)

3.3. Physical properties of hydrophobins

Besides helping in the fungi growth and colonization, hydrophobins show other interesting properties:

- Hydrophobins form multimers when they are dissolved at low concentrations, Due to the amphiphilic nature of this proteins several kind of multimers can be formed when they are in a solution, for example amphiphilic dimmers are formed when two monomers bound themselves without shielding their hydrophobic parts, in turn hydrophilic dimers can also be formed when two monomers bound together by their hydrophobic parts. These dimmers can bind together and form polymers (fibers)(fig. 3). The high number of disulfide bridges produces high stability on hydrophobins; they also tolerate temperature and pH changes.

- Due to their foaming ability, hydrophobins cause beer gushing, this phenomena could be produced for class I and class II, however only class II hydrophobins cause beer gushing (Linder et al., 2005)

- Hydrophobins can change the nature of the surfaces, turning the hydrophobic ones into hydrophilic (Teflon) this could be very useful in medical application making the pathogenic bacteria adherence harder on catheters surface and encouraged the binding of fibroblast to transplants encouraged (Scholtmeijer et al., 2001)(Zampieri et al.,2010) (Scholtmeijer et al., 2002).

- When a membrane of hydrophobins coat a surface, the surface tension decreased, this change will be bigger in air/water interfaces than liquid/liquid Interfaces (Lumdson et al., 2005).

- Hydrophobins can act as surfactants and emulsifiers.- Hydrophobins could immobilize enzymes on hydrophobic surfaces, protecting them

to temperature damage and maintaining the activity of the immobilized enzyme. e.g. Hydrophobins and EGI (endoglucanase).

- Hydrophobins could interact with other Protein-protein interaction columns, duet to hydrophobins form monolayers and other proteins could attach to them for ion exchange interactions, pH mediate the strengthens of the binding (Wang et al., 2010)

4. Production of Hydrophobines

The production of hydrophobines have been a triggering step into the production at major scale. There are several works between homologous and heterelogous methods to enhance the production of this proteins, however the yield of production have been rather low, depending the microorganism and the method the amount of produced protein vary from 60mg/L (Askolin et al., 1997) for SC3 gene from S. commune, to 600 mg/L HFBII hydrophobin from T. reesei (Hektor & Scholtmeijer, 2005).

4.1. Homologous Methods

4.1.1. Class I production

The SC3 Hydrophobin from S. commune was observed for first time by Wessels et al., (1991), this gene remain silent in young cultures but to the extent that fungi is confronted with air/medium interface the gene is activated (Wosten &Wessels, 1997). SC3 gene encodes for a class I hydrophobin. This hydrophobin is the most well characterized with EAS hydrophobin from N. crassa from class I family. Most of the functions, basic structure, and multimerization patterns for class I are based on observations from these two hydrophobins (Van wetter et al., 2000). After the discovery of SC3 gene genetic engineering techniques was used to enhance the amount of protein expressed naturally, Schuurs et al, (1997) added extra copies of the SC3 gene on a wild type strain of S.commune to enhance the production, however, both genes the endogenous gene and the introduced was silenced for methylation of the r-DNA from the introduced gene and the accumulation of transcripts in the cell. As well six hydrophobins from C. fulvum was silenced by the same posttranslational effect (Lacroix & Spanu 2009). Despite this gene silencing S. commune is the most productive fungi for class I expression (Wösten &de vocht, 2000).

Other important Basidiomycetes are Grifola frondosa an edible mushroom which encoded several class I hydrophobins (Yu et al., 2008) and Pleurotus ostreatus, the latter expresses several hydrophobins during different stages of its development e.g. FbH1 fruit-body specific hydrophobin (Armenante, 2010), Vmh3 hydrophobin which is naturally glicosilated during the vegetative growth (Sigridur et al., 1998)(Armenante, 2010)some hydrophobins could interact with glucans when they are on the medium these glucans make hydrophobins more soluble in solution (Armenante et al.,2010) (Peñas et al., 1998).

Basically the method for hydrophobin production is growing the fungi on a semi solid basic medium (S. commune minimum media, PDA agar) and then passed to a liquid minimum broth, the time of the fermentation could vary from 24 to 120 hours depending of the objective of the study (Wang et al., 2010). Most of the hydrophobin produced is cell wall bound, so for homologous methods the separation technique is a crucial step for a successful recovery (Mustahlati et al., 2011)

4.1.2. Class II methods

The ascomycetes, Aspergillus fumigatus with RodA (Class I) and RodB hydrophobins, and Trichoderma reesei HFBI and HFBII (class II), T. reesei is the most used strain to produce this protein due to their ability to secrete large amouns of of hydrolityc enzymes and the stability of its hydrophobins during the folding stage In vitro(Nakari-Setala & Pentillä. 1995). The highest production for a class II hydrophobin was made in T. reesei by the creation of an overproductive strain, adding two exttra copies of the HFB1 gene into a wild strain, HFBI copies were inserted into the cbh1 (Celobiohidrolase) promoter which is a strong producer of extracellular proteins (hydrolases) (Penttilä et al., 1987). By this system of overproduction the amount of hydrohobin was 0,6 g/L (Askolin et al., 2001). Class II have always presented higher yields than class I hydrophobins, possibly due to the chaperones and foldases are less well prepared in class I fungi strains (Hektor & Scholtmeijer, 2005).

Hydrophobin fusion technology is another technique used to produce hydrophobins, consisting in the expression of two or more genes which originally coded for separate proteins, the “fusion gene” express then a single polypeptide, this protein complexes are expressed intracellularly, they are binded to the endoplasmic reticulum as an inclusion bodies, toxicity caused by over accumulation of proteins are reduced by the formation of inert storage organelles inside the cell, thus, improving the recovery of the protein of interest (Mustalahti et al.,2011).

Despite the large number of fungi that could produce hydrophobins the best results at laboratory scale were obtained with Trichoderma reesei (Schmoll et al 2010) and Pleurotus ostreatus (Armenante, 2010).

4.2. Heterologous methods

4.2.1. Prokaryotic expression

Due to low yields and contamination problems another microorganism used for expression of hydrophobins is E. coli. Wang et al (2010) insert the HGF1 hydrophobin gene of Grifola frondosa to into the pET-28a plasmid which overexpress proteins, the efficiency of E.coli was about 100µg/100mL., Kirkland et al, (2011) used E. coli to express mHyd2 hydrophobin from the entomopathogenic fungi Beauveria bassiana which is used commercially for the biologically control of insect pests, in this research the idea was to produce an active hydrophobin on E.coli, the inserted plasmid was induced with the

addition of isopropyl-β-D-1-thiogalactopiranoside (IPTG), the correct folding and the activity of the protein was tested by drop surface transfer and contact angle measurement tests. The native hydrophobin structure was unsuccessfully cloned, the C-terminal sequence was changed in the last fifteen amino acids to get a correct folding. In the attempt to describe the mechanism of rodlet assembly Kwan et al., (2006) produce in E. coli as inclusion bodies the class I (EAS) hydrophobin from Neurospora crassa, due to the disordered structure of class I hydrophobins and its tendency to form rodlets, it was used direct mutagenesis in E. coli to probe that the mutant hydrophobin has the capacity to form rodlets as the native.. An important clinic pathogen Aspergillus fumigatus produce several hydrophobins, paris et al., (2003) identified two of them to elucidate the mechanism of RodA in the resistance against machophague attack, Schmoll et al., (2010) used T. reesei, to express DowA class I hydrophobin from A. fumigatus with the promoter of HFbII from T.reesei showing its dependency from a promoter to the expression of a hydrophobin.

FIG 4.Examples of two plasmid used during transformation of hosts for heterologous protein expression. Upper photo: AoX plasmid for Pichia pastoris, expression is activated with methanol as only carbon source (Boer et al., 2000). Lower image: Plasmids PET-33 which is activated with IPTG (isopropyl-β-D-1-thiogalactopiranoside) as only carbon source. Proteins are expressed mostly as a inclusion bodies (Armenante, 2010)(Peñas et al, 1998).

The chemical company BASF obtained two class I hydrophobins, on a pilot scale using fusion protein technology, the DewA-His6 hydrophobin from A. nidulans attached to the yaaD protein from B. subtilis this two sequences were finally cloned and produced in E.

coli, the resulted proteins were successfully formed and active, compared with SC3 and HFBII hydrophobins as blanks (Wohlleben et al., 2010).

4.2.2. Eukaryotic expression

Albuquerque et al. (2004) studied the behavior of two hydrophobins of Paracoccicoides brasilensis in paracoccidioidomycosis a relevant lung disease, these hydrophobins (Pbhyd1 and Pbyhd2) seems to be necessary during the transition of the mycellial phase. Fusarium culmorum hydrophobin (FcHyd5p) was inserted in the methylotrophic yeast Pichia pastoris, using its AOX1 promotor which is activated by comsuption of methanol as a sole carbon source (Stübner et al., 2010). Stübner et al (2010), uses Pichia pastoris to express the hydrophobin FcHyd5p from Fusarium culmorum, a fungi that causes beer gushing. Expression and transformation of P. pastoris was made PCR for propagation of the recombinant material and electroporation to induce the transformation of the cells.

Plasmids are widely used as templates for the genetic material there are many examples of plasmids, there are heterologous methods with different fungi and different host. T. reesei was transformed to overexpress and underexpress HFBII using the strain Rut-30, the proteins were overexpressed using the promoter of the celobiohydrolase which is a strong promoter. The yield of this fermentation was between 20- 300 mg/L (Bailey et al., 2002)

4.2.2.1 Plants and insects production

Heterologous proteins expression in superior organisms like plants have been used successfully with a sequence of and HFBI hydrophobin from T. reesei, Joensuu et al (2010), through infiltration of Agrobacterium tumefaciens in Nicotiana benthamiana, the hydrophobin was used to produce a green fluorescent protein and to prevent the necrosis of leaves, the hydrophobin was used too to separate the GDP with a aqueous two-phase system (ATPS) system these technology increases the production levels of the protein and the purification procedure is easier. T. reesei Hydrophobin HFBI was replicated on baculovirus to infect insect cell as a fusion protein with Avidin (an egg protein), the protein was successfully expressed on the insect cells, the yield of the infection after extraction for pure protein was 70 mg/L (Lahtinen et al., 2008)

5. Extraction and Purification

For laboratory scale hydrophobins are easily recovered by sonication of the fungi cell and centrifugation cycles to get a crude extract of the hydrophobin, these crude extracts may content cellular material for more purity, purification columns are necessary, SDS-PAGE is used to view the purity of the hydrophobin and reverse-phase high performance liquid chromatography to achieve high purity(Paris et al.,2003). Electroelution a method based in separation by applying a negative current into a electrophoresis gel causing the migration of the interest protein to the surface of the gel, this method is easy and provide high efficiency on the recovery yield (Kirkland et al, 2010) Another hydrophobins are capable of altering the the hydrophobicity of their Stübner et al (2010) uses electroelution and ultracentrifuge to purified the protein, no data about the yield of the experiment, Kirkland et

al (2010) uses electroilution to purify the protein with ph changes to prevent the folding, yield of the production about 200 to 260 µg/mL of elution fraction the total yield of purified protein was 7-10 µg/L. phague display technology was used to cloning and produce a hydrophobin from B. bassiana, using the plasmid T7 select 10-3 and carbohydrate derived coated beads to separate the phague with the protein of interest, no yield showed(Cho et al., 2007). Methanol/chloroform precipitation, centrifugation cycles, resuspension of the protein aggregate in TFA and SDS-PAGE and western blot analysis, using the promoter the yield after purification was 30 mg/L (15% of total secreted protein), Kg/L using E. coli in BASF (Wohlleben et al., 2010). Other separation method to purify proteins is Aqueous micelliar two-phase separation (AMTPS)(Lahtinen et al., 2008), folding and refolding of the protein to prevent loses. Hydrophobins are commonly attached to cell wall, this feature makes protein recuperation difficult, the amount of cell wall bind hydrophobin could arise to 85% of the total protein produced (muslalahti et al., 2011), protein extraction could be enhanced with the use of detergents like SDS and protease inhibitors to prevent the proteolysis (Askolin et al, 2001).

Only Peñas et al (2002) give some data about the yield of the hydrophobins liberation with a maximum of 21 mg/500mL.

At laboratory scale the recuperation and purification of these proteins is made using vaiorus techniques, purification columns

Fig 3. Right:Scheme of self assemble hydrophobin in solution without interfaces (Wohlleben et al.,

2010), left: aqueous micellar two phase separation micelles example(Liu et al, 1996).

6. Conclusion