Performance Hydrophobin Surfactant Proteins Using a Dual-Domain Fusion Strategy

Home , Hydrophobin

Design and Production of High- Performance Hydrophobin Surfactant Proteins Using a Dual-Domain Fusion Strategy

A THESIS

SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL

OF THE UNIVERSITY OF MINNESOTA

Natalia Calixto Mancipe

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

Ping Wang, Ph.D.

January 2019

Acknowledgements

This journey started within the concept of “biologically enhanced” or “smart” materials which combines the functionalities of proteins or whole organisms with materials commonly used in commercial goods. Dr. Ping Wang and I discussed the production of

“capturing surfaces” with enhanced affinity for specific targets, which directed us to hydrophobins. Due to their potential applications for my work and other research in our group, we opted to focus on this protein family, decision that guided the rest of my story and opened new paths that are now being continued by Benjamin Frigo-Vaz and Ying

Lei.

Many people were involved in making this possible. First, I would like to thank my adviser, Dr. Ping Wang for the opportunity of coming to the University of Minnesota, the

BBE Department and working in his lab. Also, for sharing his approach to problems with discipline, not getting too excited too soon, building up patiently and with thirst for proof to support my twinkly-twinkly and ever exciting ideas. While working with him I learnt technical and communications skills, I enjoyed peaking-in different branches of science and run head-first onto a couple of brick walls, which of course taught me many lessons that I will be working on for many more years. Thanks to Jeff, Xi, Kanghee, Sareh, Ying and Ben, friends and lab mates.

The development of this story would not have been possible without the expertise of Dr.

Brett Barney who generously gave his time and resources to help with the hydrophobin production. Special thanks to Carolann Knutson and Kalene Mulliner, from his lab, for transferring their know how. Dr. Michael Freeman was also involved in this task, I want to thank him for all his technical advice, moral support and highly appreciated caffeine input and to his student, Marissa Quijano, for propelling me to find help in a key moment of the project (the first brick wall).

From the fungal side, I would like to thank Dr. Jonathan Schilling for introducing me to this topic and giving a foundation to start looking at these important organisms. Also, for his interest in student wellbeing and for facilitating fun and engaging working environments. Thanks to Gerit Bethke from the Department of Agronomy and Plant

Genetics for providing the initial tips on Botrytis that got me started, and to Kanghee Yon for his willingness and all the spores he counted.

The passion for his work and expertise of Dr. Greg Haugstad, from the Characterization

Facility at the University of Minnesota, are contagious, I was lucky to have the opportunity to learn from him during the final stages of the experimental part.

I also want to thank the Department of Bioproducts and Biosystems Engineering and

MnDRIVE for their financial support, specially to Dr. Shri Ramaswamy for his efforts towards the funding and wellbeing of graduate students as well as our new Department

Head Dr. Gary Sands.

This work shows that “nothing gets done without effort” but with it, and many good people, good things can happen. So, I want to thank my friends Pablo, Julia and Aritra for the fun moments, your affection, support and companionship; and to Sarra Beckman-

Chasnoff and all the Grief Group members for their support, hey made a night and day difference in my experience.

Finally, an enormous thankyou to my family, for bearing with me through ups and downs and giving so much along the way: to my parents for their example and for giving me a foundation in love and faith, to Troy and Glorita for adopting me and easing my way into the American culture and Gila for being an angel when I needed her the most. Also, to my sister, my always-caring Chichipersona, my uncle Ruben, checking on me every time

I am wandering through places, and my sweet Lalo.

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Dedication

Para mi papá, Luis Francisco Calixto, porque con su ejemplo me mostró la importancia del trabajo honesto, lo divertido que son el ingenio y la imaginación, por no darse por vencido y por darnos tantísimo amor “hasta la muerte y más allá”. Espero no olvidar sus enseñanzas de fuerza, nobleza y generosidad. Para Diosito y la Virgensita, porque la fe y su compañía son la mejor herencia que tengo.

Abstract

Biosurfactants are amphipathic molecules required for vital processes in all life forms.

They help to reduce surface tension facilitating interfacial processes such as breathing and evaporative cooling, modulate environmental conditions, control surface wettability, interact with substrates, form protective layers, etc. Thus, nature has evolved a wide variety of biosurfactants combining hydrophilic building blocks such as acid groups, sugars or polar amino acids with hydrophobic ones such as lipids, creating an enormous pallet of possibilities. Among them, hydrophobins (hereafter HFBs) are a family of self- assembling surfactant proteins with the highest surface activity known to date and an intrinsic amphipathic structure that does not require additional functional groups such as lipids or sugars. These characteristics give them a great potential for their industrial use as interfacial stabilizers, dispersal agents, surface modifiers and molecular anchors for protein immobilization on solids.

The unique sequences and folding structures of HFBs particularly promote interfacial and intermolecular interactions, along with robust self-assembling mechanisms and surface activity. Unfortunately, a lack of knowledge on their sequence-structure-function relationships hinders the optimal selection of proteins for specific applications as well as manipulation of their properties. For a specific consideration, the use of HFBs for standard coating processes usually results in irregular products; at the same time, such an

application requires a large-scale production that has been difficult to achieve limited by the productivity of native organisms, the HFB toxicity for heterologous hosts and the challenges of their purification. These obstacles suggest the need to leverage HFBs’ interfacial behaviors to better fit a broader range of applications.

Therefore, an HFB from the fungus Trichoderma reesei is taken as a model surfactant protein in this work to explore the variation of its characteristics by a modular fusion strategy, as an alternative approach to protein directed mutagenesis and engineering. We aim to combine its high surfactant activity with the functionalities of different fusion partners to enhance its productivity and interfacial properties.

Our results show that the fusion with a small metal binding protein from Nitrosomonas eurepaea (SMBP) achieves much improved product solubility and easier purification without compromising the hydrophobin properties. SMBP-HFBII shows a critical micelle concentration (CMC) < 0.5 mg/l and is prone to form stable nanobubbles of 50-60 nm radius and thin films at liquid-air interfaces. The adsorption of SMBP-HFBII on hydrophilic substrates (mica and glass) generates homogeneous coatings that reverse their wettability increasing the water contact angle (WCA) 460% and 70%, respectively.

Adsorbed SMBP-HFBII also demonstrated a slight increase of Botrytis cinerea spores’ adhesion to coated glass, dependent on pH and spore concentration.

This work shows that fusion HFBs can expand the application potentials of their native parent proteins as the fusion provides a powerful avenue to manipulate their functionalities. The lack of knowledge on their structure-function relationships and

uncontrolled self-assembling can be subsidized by the addition of domains that influence the overall protein performance. This fusion strategy also enhances HFB productivity, facilitating their use in research and industry.

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Table of Contents

List of tables ...... x List of figures ...... xi List of abbreviations ...... xiii Chapter 1: Introduction to biosurfactants: biology, biochemistry and applications ...... 1 1.1 Water surface tension and the role of biosurfactants ...... 1 1.2 The diversity of surfactant proteins ...... 3 1.3 Fungal hydrophobins: a highly-active family of surfactant proteins ...... 6 Chapter 2: Improved solubility of a dual-domain hydrophobin fusion with a highly hydrophilic metalloprotein partner ...... 14 2.1 Abstract ...... 14 2.2 Introduction ...... 15 2.3 Materials and methods ...... 17 2.3.1 Materials and microbial strains ...... 17 2.3.2 Design of HFBII fusion proteins ...... 17 2.3.3 Establishment of growth conditions for protein production ...... 19 2.3.4 Protein production and purification ...... 20 2.4 Results ...... 21 2.4.1 Design and production of HFBII fusion proteins ...... 21 2.4.2 Purification of SMBP-HFBII and GFP-HFBII by liquid chromatography ...... 28 2.5 Discussion...... 29 Chapter 3: Taking advantage of a polarity shift: interfacial and self-assembling behaviors of hydrophobin fusion proteins ...... 34 3.1 Abstract ...... 34 3.2 Introduction ...... 35 3.3 Materials and Methods ...... 37 3.3.1 Materials ...... 37 3.3.1 Assessment of surfactant behavior by light and fluorescence microscopy ...... 37 3.3.2 Protein deposition on solid substrates ...... 38 3.3.3 Analysis of protein interfacial assemblies by AFM ...... 40 3.3.4 Analysis of surface chemical composition by XPS ...... 41

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3.3.5 Surface wettability measurements ...... 41 3.4 Results ...... 42 3.4.1 Interfacial activity and foaming effect of HFBII fusion proteins ...... 42 3.4.2 Protein self-assembled micro and nanostructures ...... 44 3.4.3 Immobilized SMBP-HFBII at the solid-liquid interface ...... 47 3.4.4 Effect of SMBP-HFBII coatings on material properties ...... 50 3.5 Discussion...... 53 Chapter 4: Biocontainment of fungal spores by SMBP-HFBII coatings ...... 59 4.1 Abstract ...... 59 4.2 Introduction ...... 59 4.3 Materials and methods ...... 62 4.3.1 Materials and microbial strains ...... 62 4.3.2 Preparation of solids substrates ...... 62 4.3.3 Botrytis cinerea growth conditions ...... 62 4.3.4 Air dispersion of spores ...... 63 4.3.5 Adhesion of spores in solution ...... 63 4.3.6 Quantification of spore adhesion by light microscopy ...... 63 4.4 Results ...... 64 4.4.1 Effect of surface hydrophobicity on spore adhesion to substrates ...... 64 4.4.2 Effect of pH and protein coating on the adhesion of spores to glass substrates ...... 65 4.4.3 Effect of spore concentration on their adhesion behavior ...... 66 4.5 Discussion...... 68 Chapter 5: Conclusions and further directions ...... 71 References ...... 74

List of tables

Table 1. Examples of biological functions performed by hydrophobins ...... 7

Table 2. Properties of natural and engineered class I and class II HFBs ...... 10

Table 3. Examples of HFB technical and industrial applications ...... 11

Table 4. Hydrophobin production systems: achievements and challenges ...... 12

Table 5. Primers for HFBII fusion proteins design ...... 18

Table 6. Protein expected parameters and origin ...... 23

Table 7. Surface relative atomic composition of SMBP-HFBII coated mica ...... 50

Table 8. Water contact angle of protein-coated materials ...... 51

List of figures

Figure 1. Reduction of water surface tension by biosurfactants ...... 2

Figure 2. Diverse structures of biosurfactants ...... 4

Figure 3. Structural characteristics of class I and class II hydrophobins...... 9

Figure 4. HFBII genomic sequence modifications ...... 22

Figure 5. Expression of the initial set of HFBII fusion proteins ...... 22

Figure 6. Differential purification efficiency of yaaD-HFBII based on buffer selection 24

Figure 7. Variable transformation efficiencies of HFBII fusion proteins ...... 25

Figure 8. Effect of temperature and SMBP-HFBII expression on the reduction of total cellular protein ...... 26

Figure 9. SDS-PAGE and fluorescence images of expressed GFP-HFBII...... 27

Figure 10. SDS-PAGE analysis of SMBP-HFBII and GFP-HFBII purifications ...... 28

Figure 11. Hydropathy characteristics of SMBP-HFBII...... 32

Figure 12. Methods used for protein deposition on solid substrates ...... 40

Figure 13. Interfacial and foaming activities of GFP-HFBII and SMBP-HFBII...... 43

Figure 14. Microstructures formed by self-assembling of GFP-HFBII stabilized foams 44

Figure 15. AFM images of SMBP-HFBII globular assemblies...... 45

Figure 16. Topography of SMBP-HFBII self-assembled film structures ...... 47

Figure 17. Surface topography and protein content of SMBP-HFBII coatings analyzed by

AFM and XPS ...... 49

Figure 18. Modulation of materials’ surface wettability by SMBP-HFBII coatings ...... 52

Figure 19. Surface protein content of different materials coated with SMBP-HFBII analyzed by XPS ...... 53

Figure 20. Hypothetical HFBII-fusion protein distribution in the proximity of the air- liquid interface ...... 56

Figure 21. Effect of material hydrophobicity on B. cinerea spore adhesion to surfaces . 64

Figure 22. Effect of pH and SMBP-HFBII coating on B. cinerea spore adhesion...... 66

Figure 23. Adhesion of spores in solution at high spore concentration ...... 67

Figure 24. Effect of spore concentration on the spore adsorption to coated and uncoated glass...... 68

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List of abbreviations

AFM Atomic force microscopy

BSA Bovine serum albumin

CBD Cellulose binding domain

CMC Critical micelle concentration

CNT Carbon nanotube

E. coli Escherichia coli

GFP Green fluorescent protein

HFB Hydrophobin

IB Inclusion body

IP Isoelectric point

LC Liquid chromatography

MBP Maltose binding protein

MW Molecular weight

N. europaea Nitrosomonas europaea

SMBP Small metal binding protein

SP Signal peptide

T. reesei Trichoderma reesei

XPS X-ray photoelectron spectroscopy

WCA Water contact angle

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Chapter 1: Introduction to biosurfactants: biology, biochemistry and applications

1.1 Water surface tension and the role of biosurfactants

Life happens in and around water, our chemical reactions and vital functions occur in water, at or through interfaces. Thus, we depend on the appropriate transfer of molecules and structures through them and the ability to modulate their physical properties. Water has high surface tension. This is because water molecules are small and highly polar, which triggers the formation of numerous hydrogen bonds resulting in attractive interactions amongst themselves. In the bulk solution, the sum of equivalent forces on a single molecule from the surrounding water results in a zero-net force. But at interfaces there is an unbalanced interaction and a net force directed towards the bulk, which causes the water surface to contract and “bead up”. This is called surface tension.

High surface tension limits the transfer of molecules and structures through interfaces.

Therefore, it is a key property that modulates vital functions that rely on mass transfer such as breathing, evaporative cooling, among many other processes that will be discussed in this chapter. Hence, organisms have evolved a variety of molecules to control surface tension, namely biosurfactants1. Biosurfactants, also known as interfacially-active or surface-active molecules, help minimize surface tension due to their amphipathic nature. Their general structure holds both hydrophilic and hydrophobic moieties that allow the simultaneous interaction with substrates of opposite polarity and

their spontaneous migration to interfaces. At hydrophobic/hydrophilic interfaces they adopt an oriented conformation with each of their moieties facing and interacting with the respective environment, reducing the unbalanced net forces, hence the surface tension

(Figure 1).

Figure 1. Reduction of water surface tension by biosurfactants Schematic diagram illustrating the attractive forces in play between molecules in a water droplet in the (left) absence and (right) presence of a surfactant. In the bulk liquid, water molecules make hydrogen bonds with neighboring water molecules in all directions, resulting in no net force. In contrast, water molecules at the edge of the drop cannot interact with air on one side and are therefore pulled inward by water molecules inside, giving rise to water surface tension. In the presence of a surfactant, the surfactant molecules migrate preferentially to the interface as they form hydrogen bonds with water molecules below them and to simultaneously form hydrophobic interactions with air molecules above. This leads to a significant reduction of the net force experienced at the interface and hence a reduction in surface tension1.

From a macromolecular perspective, biosurfactants are essential for the modulation of interfacial properties like surface hydrophobicity, formation of boundary structures such as membranes or coatings, as well, as regulation of important environmental interactions like attachment, cellular recognition or cross-membrane mass transfer. There is a wide variety of biosurfactants in nature, resulting from the combination of hydrophilic building blocks such as acids, sugars or polar amino acids with hydrophobic ones such as lipids or

aliphatic amino acids. Some common examples of biosurfactants include fatty acids, phospholipids, lipopeptides, glycolipids, and glycoproteins.

Some of the most extensively studied biosurfactants belong to the glycolipids group, specially rhamnolipids which have mono or disaccharides combined with long-chain aliphatic acids, also the lipopeptides and amphipathic proteins (Figure 2). Their high surface activity, biocompatibility, and biodegradation potential has spurred applications in several industries as emulsifiers or foam stabilizers (cosmetics and food preparations), dispersal agents for hydrophobic compounds in aqueous systems (drug delivery, bioremediation, detergents), as antimicrobials, among others. Therefore, biosurfactants are continuously explored and engineered with respect to cost-effective production and feasibility for applications in the biomedical, food, cosmetics, oil and new materials industries2,3.

1.2 The diversity of surfactant proteins

Surface-active proteins have diverse functions and structures in nature and can be found in a variety of organisms ranging from bacteria to fungi, plants and animals. As any other protein, they are composed by a chain of charged, polar and non-polar amino acids which give them an intrinsic amphipathic potential. The actual amphipathic character depends on their primary sequence, 3D folding, conformational stability, quaternary structure and posttranslational modifications. In turn, these endogenous features are influenced by environmental factors such as pH, temperature, ionic strength, and presence of binding partners and interfaces1,4. 3

Figure 2. Diverse structures of biosurfactants Three examples of amphipathic structures are shown: a monorhamnolipid (A), the surfactin lipopeptide (B) and the MPGI hydrophobin (C). All structures have a polar or charged portion and a hydrophobic part. In C charged residues are shown in blue and red and the hydrophobic ones in grey.

Representative examples of surface-active proteins in bacteria are the small lipopeptides produced by non-ribosomal peptide synthetases (NRPSs)5,6. Their general structure consists on a cyclic peptide backbone linked to a fatty acid chain, which gives the molecule its amphipathic character. One of the best studied examples is the surfactin from Bacillus subtilis (Figure 2B), which has a CMC in the order of 50 – 1.4 mg/L depending on the media7,8 and can reduce water surface tension from 72 to 27 mN/m at a

concentration of 0.005% (0.5 mg/L)1. This molecule has been related to a variety of functions ranging from facilitating bacterial dispersion to lytic and inhibitory activities against viruses, other bacteria and fungi through the formation of transient pores in outer membranes1.

Other examples of bacterial surfactant proteins are the phenol-soluble modulins (PSMs), from Staphylococcus aureus, rodlins and chaplins in Streptomyces species, phasins, and biofilm surface layer proteins (BslA) from B. subtilis also known as “bacterial hydrophobins”1,4. These proteins play important roles in bacterial life cycle. For instance,

PSMs are involved in biofilm formation, dissemination, and have cytolytic activity.

Phasins, stabilize cytosolic polyhydroxyalkanoate (PHA) storage grains. Rodlins and chaplins coat aerial hyphae and spores facilitating air-dispersion. BslA forms amphipathic layers over B. subtilis biofilms protecting them from wetting and gas penetration9,10.

Surfactant proteins from plants such as soy globulins have been long used in the food industry to stabilize emulsions, but their specific functionalities haven’t been systematically studied. Similarly, complex amphipathic mixtures composed of lignin, lipids and proteins have been shown to play an important role in nutrient transfer through the xylem by modulating surface tension and bubble formation, but further molecular characterization is required11,12.

Examples of biosurfactants in animals include frogs’ ranaspumins, horses’ latherin, and mammals’ SPLUNC1, pulmonary surfactant proteins. Ranaspumins undergo a conformational change at the air-water interphase and create stable foam nests for the 5

development of fertilized eggs. Likewise, latherin suffers a conformational change at hydrophilic/hydrophobic interphases, modifying the wettability of horse hair which allows a rapid spread of sweat and effective evaporative cooling. In mammals, SPLUNC1 inhibits growth of gram-negative bacteria in the upper respiratory tract, pulmonary surfactant proteins help reduce surface tension of the alveolae, hence preventing collapse when breathing, and casein micelles act as nanocarriers for calcium phosphate in milk1.

In fungi, important examples of surface-active proteins are the cerato-platanins, repellents and hydrophobins. Cerato-platanins are carbohydrate-binding proteins thought to mediate interaction with plants and the hyphal growth of submerged mycelia. Repellents are small peptides (30 - 55 residues) important for the development of aerial structures.

Hydrophobins (HFBs) are a protein family ubiquitous in filamentous fungi with low sequence similarity, a conserved 3D structure and a strong amphipathic character; they have a key role in fungal physiology and pathogenicity and have been the center of extensive research for their technological potential13,14.

1.3 Fungal hydrophobins: a highly-active family of surfactant proteins

Fungal HFBs constitute a protein family with unique surfactant and self-assembling characteristics and the highest surface activity known to date. They were first described in the early 90s amid studies of highly expressed genes in filamentous fungi15,16, and later were shown to have complex and varied regulatory systems for gene expression. Several studies have shown that their expression is modulated by internal factors such as the specific stage on the fungal life cycle17 and morphology (spores, fruiting bodies, 6

mycelia)18 as well as environmental conditions like light17, carbon source or media type

(liquid/solid)19.

HFBs play vital roles in fungal physiology and pathogenicity as evidenced by the diversity of functions that depend on them (Table 1). These include coating/protective actions such as the formation of external layers, the adhesion to surfaces important in host-pathogen interaction and infection, and the modification of surface/interface properties for example lowering water tension to facilitate the growth of aerial structures20.

Table 1. Examples of biological functions performed by hydrophobins Organism Protein Function Botrytis cinerea Bhp1, Bhp2, Required for fruiting body formation and morphology18 Bhp3 BcHpb1 Modulates the adhesion to surfaces21 Clonostachys rosea Hyd1, Hyd3 Coats conidia, gives them their hydrophobicity22 Aspergillus oryzae RolA Increases adhesion of lytic enzymes to hydrophobic carbon sources23 Cladosporium fulvium HCf-6 Required for adhesion to host during infection24 Trichoderma HFB2-6 Interaction with plant tissues25 asperellum

HFBs are classified as Class I or II depending on their hydropathy plots26,† (Figure 3), the stability of their macromolecular assemblies, their 3D structure and the spacing of 8 conserved cysteine (Cys) residues on their primary sequence. Both classes are produced

† Hydropathy plots show the summed hydrophilicity of a peptide (Y coordinate), based on a scale of the hydrophobic and hydrophilic properties of the common 20 proteinogenic amino acids, as a function of the aminoacid position in the sequence (X coordinate), thus giving information of the hydropathic character of a protein26. 7

by Ascomycetes while Basidiomycetes‡ only produce Class I13. Class I HFBs are bigger proteins (150 - 300 residues) with a flexible 3D structure that allows a conformational change required for their self-assembling. Their self-assembled structures are more stable than those of Class II, which require the use of strong acids and high temperatures to break. These structures are often described as “rodlets” that reach micrometer-size lengths27. At the air-water interface rodlets are compressed and produce multilayered membranes that have been shown to reduce water tension28.

Compared to Class I, Class II HFBs are smaller (70 – 100 residues) and have compact and rigid structures that do not undergo a conformational change prior to multimerization or self-assembling. As a result, they also show faster self-assembling responses29.

Typically, Class II HFBs do not produce rodlets and at the air-water interface they form elastic monolayers instead of rigid multilayers. These structures can be easily disassembled by detergents and heat.

The HFB family has low sequence similarity, even within the same organism, but a highly conserved amphiphilic tertiary structure that drives their surfactant behavior. All

Class II HFBs have a small (5-10 kDa), robust, and globular folding. The typical example is formed by one β-barrel, one α-helix and four loops, held together by 4 disulfide bridges30 (Figure 3A). The 8 Cys residues involved in those bonds are present in all HFBs and show a characteristic spacing pattern: Cys 2 and 3, as well as 6 and 7, are next to each other while the rest do not have others as near neighbors13.

‡ Most of the known fungal species are basidiomycetes which include all yeasts, and unlike ascomycetes, basidiomycetes only have sexual reproduction. 8

Figure 3. Structural characteristics of class I and class II hydrophobins. A: Crystal structures, charge and hydrophobicity distribution of DewA, a class I hydrophobin (left) and HFBII, a class II hydrophobin (right). B: Corresponding hydropathy plots. Hydrophobic residues have higher scores, hydrophilic residues have negative values. Note the smaller size, more compact folding, and different hydrophobicity distribution of HFBII as a representative of its class. Molecular models were prepared using entries 2SLH and 2B97 from the Protein Data Bank and Discovery Studio software, hydropathy plots were prepared using the ProtScale tool from ExPASy31.

Their folding generates stable amphiphilic structures with a patch of hydrophobic residues exposed to the aqueous environment, opposite to a cluster of charged and polar amino acids. Therefore, their amphipathic character does not require complex posttranslational modifications like the addition of lipids or sugars nor conformational changes32,33. The existence of the hydrophobic patch drives the spontaneous formation of soluble oligomers in aqueous solution or the migration to interfaces where they self- assemble in organized structures that vary in size, shape and mechanical properties. Once

formed, these structures can decrease the water tension and change the wettability of surfaces34.

HFB self-assembly mechanism is not clear yet and could be different for different proteins. It has been suggested that hydrophobic interactions drive the formation of small oligomers in solution, in a concentration-dependent manner, being able to form bigger assemblies such as fibers35. They also migrate to hydrophilic/hydrophobic interfaces where they reassemble forming films (at the air-water boundary)36, or adhere to solid surfaces (solid-liquid boundary) generating coatings with different biophysical properties

(Table 2). Class I HFBs form resistant multilayered membranes at the air-water interface that, after drying or compressing, evolve to insoluble rodlet-like micro-structures. Class II

HFBs create flexible monolayers and weaker fibril-like aggregates. Class I HFBs also show a stronger adhesion to solid surfaces than Class II34.

Table 2. Properties of natural and engineered class I and class II HFBs Hydrophobin Fungus Surface Hydrophilic Hydrophobic Rodlets activity side WCA side WCA (mJ m-2) Class I SC3 S. commune 27-32 36 ± 3 115 ± 12 yes Deglycosylated S. commune 32 66 ± 6 ND ND SC3a RGD-SC3 S. commune 32 44 ± 2 122 ± 4 yes TrSC3 S. commune 32 73 ± 3 119 ± 3 yes ABHI A. bisporus ND 63 ± 8 113 ± 4 yes ABH3 A. bisporus 37 59 ± 5 117 ± 3 yes HGFIb G. frondosa 45 62 ± 2.5 ND yes Class II HFBI T. reesei 42 59 ± 1.3 60 - 64 no HFBI T. reesei 35 - 60 - 70 no

CRP C. parasitica 32 22 ± 2 ≥ 90c no CFTH1 C. fusiformis 33 60 ± 5 105 ± 2 no Surface activity measurements and coatings were performed at 100 μg·mL−1 unless mentioned otherwise. ND, not determined; a22 μg·mL−1; b80 μg·mL−1; ccoating not homogenous. Adapted from Zampieri et al. 201029.

HFB high interfacial activity, surfactant behavior, self-assembling capabilities and robust structures, have made them great candidates for several technical applications in various fields (Table 3). Unfortunately, a lack of knowledge about their sequence-structure- function relationships has limited the optimal selection of proteins for specific applications as well as an efficient manipulation of their properties.

Table 3. Examples of HFB technical and industrial applications Protein Application HFBII foam stabilizers37,38 HFBI dispersion and delivery of hydrophobic-compounds39 or carbon nanotubes40 DewA, HFBI tags for protein immobilization on solids41–43 for biosensors and biochips Isolated from Patterning agents for silicon micromachining processes44 Plerotus ostreatus Hyd2 modify the wettability of surfaces45 DewA enzyme-recruiting systems to stimulate their activity on hydrophobic materials and biodegradation capabilities42 HFBI protein purification tags via ATPS46–49 HGFI Dispersion of enzymes in hydrophobic substrates50 SC3 Production of low-friction surfaces51 SC3 Promote cell adhesion and growth in culture52

The use of native HFBs often leads to uncontrolled foaming behaviors and unpredictable distribution of proteins when immobilized on solid substrates, which is a challenge for the use of standard purification techniques and protein deposition methods53–56.

Additionally, large-scale production of HFBs has often been reported to be associated 11

with low yield of protein25, the formation of inclusion bodies22–24 or silencing issues26

(Table 4). Hence, engineering approaches are required to control HFBs’ solution, self- assembling and interfacial behaviors as well as production schemes that enhance their productivity.

Consequently, there have been numerous attempts to improve the production and use of

HFB through process development and protein engineering. The genetic manipulation of the native producers, the optimization of their growth conditions or the modification of

HFB genes and heterologous expression have been explored to considerable success

(Table 4)19. Nonetheless, the stability of the expression systems, product solubility and later purification are still challenging. Similarly, there have been encouraging reports on the modulation of HFBs’ interfacial properties by site directed mutagenesis or deletion of whole gene fragments, demonstrating the potential use of such approaches to modify their native characteristics and improve their behavior and applicability52,60.

Table 4. Hydrophobin production systems: achievements and challenges Host Organism Protein Class Yield (mg/L) Notes T. reesei HFBI, HFBII II 25-30 (wild type) T. reesei HFBI, HFBII II 240 Multiple copies of hfb genes, protein secreted to the culture media or recovered from spores61 T. reesei HFBII II 40x103 Biofilm reactor19, secreted to the media, purified by ATPS Saccharomyces HFBII II 260 Multiple copies of hfb2 gene38, purified cerevisiae by foam fractionation Escherichia coli mHyd2 I 7-10 Intein-mHyd2 fusion protein in IBs, Rosetta2 (DE3) purified by LC45 Escherichia coli DewA I ~103 yaaD-DewA fusion proteins as IBs, purified by LC57

Nicotiana benthamiana HFBI-VI Not reported IBs, purified by ATPS47 HYD3-5 IBs, purified by ATPS46

Picchia pastoris HGFI I 300 Controlled O2 and methanol concentration conditions62

This work explores the use of a fusion strategy to enhance the native properties of HBFs by the production of dual-domain surfactant proteins. Specifically, we investigate several fusion partners aiming to modify HFBs’ hydropathic characteristics to influence its solubility and interfacial behaviors. Our results show that the HFB fusion with a small metalloprotein from Nitrosomonas europeaea (SMBP)63 increases its solubility, facilitates its purification and enhances its affinity for hydrophilic surfaces. It was further demonstrated that the fusion HFBs can be applied to improve the coating process, while conserving its native surfactant and self-assembling characteristics (CMC < 0.5 mg/l).

We envision the use of this strategy to enhance the properties of many other HFBs, helping to create a new palette of surfactant proteins better fit for our technical resources and desired applications.

Chapter 2: Improved solubility of a dual-domain hydrophobin fusion with a highly hydrophilic metalloprotein partner

2.1 Abstract

Hydrophobins (HFBs) are a family of interfacially active proteins that are of interest for a variety of applications. Native HFBs are proven to be difficult to produce and purify due to their inherent high surface activity and self-assembling behaviors. Genetic manipulation of native producers and heterologous hosts typically yields low amounts of product, usually in inclusion bodies and silencing issues. Therefore, improved systems that enable a more viable production of functional HFBs and HFB fusion proteins are required.

In our work we explore the production of dual-domain HFB fusion proteins to improve productivity, with a focus on solubility and recovery. Our results indicate that a small metal binding protein from Nitrosomonas europaea (SMBP) improves solubility of the fusion HFB and allows its recovery using standard metal affinity liquid chromatography.

The effective modulation of HFBs’ solubility and ease of purification is particular appealing to use this strategy as a general method to improve the production of HFB and

HFB fusion proteins.

2.2 Introduction

HFBs are the most surface-active proteins known to date and with unique self-assembling capabilities. Native HFBs are produced by filamentous fungi at mg/l concentrations and secreted to the media13. Their production is regulated by numerous environmental and endogenous factors such as light, carbon source, media type, fungal age, etc. The low yields and slow growth of native producers call for engineering approaches for a large scale production and purification of HFBs14. In this work we explore a fusion strategy to enhance the solubility and facilitate the recovery of HFBs.

Previous research on improved production systems has explored the use of heterologous hosts, protein engineering and tailored downstream processing approaches14. Currently,

Unilever in collaboration with VTT Technological Research Centre of Finland and BASF are the main hydrophobin suppliers. VTT-Unilever uses an overproducing strain of T. reesei for HFBI and HFBII, class II hydrophobins that are later purified from the culture media by ATPS followed by HPLC. BASF produces proteins H*A and H*B in E. coli.

These proteins are recombinant class I HFBs (DewA) that include a N-terminal sequence added for stabilization and higher yields57. They are produced as inclusion bodies and then purified by Ni+ affinity LC with a 65% purity14.

19 Alternative production approaches such as biofilm reactors , CO2 foaming as a pre- purification step64 and the use of heterologous hosts such as yeast38, bacteria22,24 and plants23 have also been explored. Biofilm reactors have reported the highest yields of soluble HFB. However, heterologous expression generally achieves low yields, the formation of inclusion bodies45,47 and silencing issues59. Furthermore, purification

processes require harsh conditions such as high temperature or the use of detergents which is not suitable for potential enzymatic fusion partners30,48,62,64,65. The limitations on the use of standard production techniques compatible with the wide range of possible hydrophobin fusion partners hampers the effective use of this protein family.

HFBII is one of the most studied HFBs and one of the few examples with a resolved crystal structure. It was discovered by heterologous gene hybridization with hfb1, and later isolated from the fungal spores by acid extraction and from the culture media by bubbling17. Structurally, it is a small globular protein of 86 amino acids, 15 form the signal peptide and 71 the mature protein. It has 7.2 kDa and an approximate diameter of 3 nm. HFBII has a regular class II HFB folding with disordered N and C termini and a flat hydrophobic patch that expands through 12% of its total surface, composed mainly by aliphatic residues (Val, Leu, Ile and Ala). HFBII only has two aromatic residues Phe8 and Phe39 that are not part of the hydrophobic patch.17,32,33 Its disordered N and C termini are exposed to the solvent and are not part of the hydrophobic patch, which makes them possible modification sites for fusion strategies66. We chose HFBII as a model HFB for our study.

Metalloproteins are a diverse family of proteins produced by bacteria to modulate their metal intake from the environment67. Structurally these proteins contain highly polar domains and a number of amino acids responsible for the interaction with the metals such as cysteines or histidines (His)68. SMBP, a small metalloprotein from Nitrosomonas europaea63, has a robust structure formed by two alpha helixes and is highly soluble because of its 27% of charged amino acids and its 16 His residues exposed to the surface.

The affinity of this protein for metal ions allows its use protein tag for purification via metal affinity LC.

We designed several HFBII fusion proteins to probe the heterologous expression and enhanced recovery of the resulting fusion proteins. Our results show that HFB fusion productivity can be modulated by the fusion partner. The fusion SMBP-HFBII could be transiently expressed in E. coli, showing improved solubility compared to the other fusions, and purified from the soluble fraction by non-denaturing metal affinity LC.

2.3 Materials and methods

2.3.1 Materials and microbial strains

Buffer salts, LB media, and HisPur Ni-NTA Ni+ resin were bought from Fisher Scientific

(Pittsburgh, PA, USA). GeneElute Plasmid Miniprep kit, lysozyme, DNase and Amicon

Ultra Centrifugal Filters 10 kDa NMWL were bought from Sigma-Aldrich (St. Louis,

MO, USA). Restriction enzymes were bought from New England Biolabs (Ipswich, MA,

USA). All other chemicals including salts and acids for buffers were purchased from suppliers of analytical or higher purity and used as received.

Escherichia coli JM109, TB1 and BL21 and BL21 A1were used for cloning and protein production.

2.3.2 Design of HFBII fusion proteins

HFBII coding sequence (P79073 (NCBI accession number), Y11894.1 (gene), 2B97

(PDB structure)) including the signal peptide (SP), was modified to exclude all introns 17

and then was codon optimized for E. coli using the Reverse Translate Tool69. This sequence (hereafter “HFBII”) was used to design six different constructs harboring

HFBII and additional purification and functional fusion partners.

The three first constructs were designed in accordance to Wohlleben et al.57 The 40 first bases of yaaD were added to the N-terminus of HFBII. yaaD is a Bacillus subtilis protein that, when expressed as a truncated N-terminal partner in E. coli, is reported to increase the yield of DewA, a class I hydrophobin from Aspergillus nidulans. The first construct included the yaaD followed by the HFBII and a C-terminus His6 tag (pNCM001). The second and third constructs had a carbon nanotube binding peptide (CNTbp70) added

71 before the His6 tag (pNCM002), and a cellulose binding domain (CBD ) added in the same place (pNCM003). All constructs were synthesized and inserted in pET28α under a

T7 promoter by Shanghai Rui Di Biological Technology Co. (Shanghai, China).

In a second stage of design, the coding sequence of HFBII including its SP was amplified by PCR using the primer sets NCM001/NCM002, NCM003/NCM002 and

NCM001/NCM004 (Table 5). Then it was digested with EcoRI/HindIII and inserted into a pUC19-derived plasmid (pBBTET3-Col1). The correct insertion was confirmed by sequencing and the resulting plasmids named pNatalia2, pNatalia3 and pNatalia4.

Table 5. Primers for HFBII fusion proteins design ID Sequence 5’ → 3’ NCM001 NNNGAATTCATGCAGTTTTTTGCGGTGGCGCTGTTTGC (F) NCM002 NNNAAGCTTAAAAGGTGCCAATCGCTTTCTGGCACAG (R) NCM003 NNNGAATTCCATGGTATGCAGTTTTTTGCGGTGGCGCTGTTTGC (F) NCM004 NNNAAGCTTAGATCTTTAAAAGGTGCCAATCGCTTTCTGGCACAG (R) F: forward primer. R: reverse primer. Underlined regions are restriction sites. 18

pNatalia2 was digested again with EcoRI/HindIII and ligated into pPCRWE258 for the expression of a C-terminal HFBII fusion of a cleavable Maltose Binding Protein (MBP,

40.7 kDa). This was named pNatalia6. Similarly, pNatalia3 was digested with

NcoI/HindIII and ligated into pPCRSMBP11 for the construction of a C-terminal HFBII fusion of a Small Metal Binding Protein (SMBP, 9.7 kDa). This was named pNatalia7.

Finally, pNatalia4 was digested with BglII/EcoRI for insertion in pNatalia5 (a modified version of pBB082, which allows C-terminal fusions) for the expression of a C-terminal

HFBII fusion of a N-terminal His6 tagged Green Fluorescent Protein (GFP, 30.3 kDa).

This was named pNatalia8. For these constructs all initial plasmids (pBBTET3-Col1, pPCRWE258, pPCRSMBP11 and pBB082) were kindly provided by Brett Barney.

E. coli JM109 was used for all plasmid manipulations. The final expression vectors

(pNatalia6-8) were purified and stored at -20°C. All final constructs were sequenced for confirmation of the full inserted region prior to transformation of the expression strains.

The expected protein parameters were calculated using Expassy ProtParam72.

2.3.3 Establishment of growth conditions for protein production pNatalia6 was transformed into E. coli TB1, pNatalia7 and pNatalia8 in E. coli BL21 and

BL21A1. Antibiotic concentration used were 50 mg/L kanamycin for pNatalia7 and pNatalia8 or 100 mg/L ampicillin for pNatalia6. For inhibition of protein expression, the media was supplemented with glucose to a final concentration of 30 mM.

After transformation with the respective plasmids, E. coli was grown overnight in 10 mL of LB supplemented with the specific antibiotic at 37°C and 150 rpm. The saturated

culture was then diluted with fresh media and the biomass yields tested with different growth and induction temperatures. Induction was done with 50 mg/L IPTG for BL21 and TB1 strains while 0.2% L-Arabinose with 50 mg/L IPTG for BL21A1. The total biomass and HFB fusions yield was estimated from SDS-PAGE analysis and used as a qualitative measure for the selection of the best production conditions for each protein.

2.3.4 Protein production and purification

LB media supplemented with 50 mg/L kanamycin or 100 mg/L ampicillin was used for cell growth. Freshly transformed cells were grown overnight in 10 ml of media, at 37°C,

230 rpm. Then the saturated culture was diluted by a ratio of 1:100 and maintained at

30°C for 8 h at 200 rpm. When the culture OD600 ~ 3, then expression was induced with

50 mg/l IPTG and maintained for 10 h at 22°C and 150 rpm.

Cells were recovered by centrifugation (7 min, 7000 rpm, 4°C). The supernatant solution was discarded, and the cell pellets were resuspended in 30 ml of lysis buffer. The lysis buffer contained 50 mM phosphate buffer, 300 mM NaCl (PBS), 0.5 mg/ml lysozyme, 1 mg/ml DNase and 1 mg/ml CHAPS; the pH was adjusted to 7.2. The cell pellets were then lysed by sonication (four 30 s pulses with 15 s delay, maintained on ice). The cell debris were separated by centrifugation (10 min, 12000 rpm) and the supernatant used for protein purification using Ni+ affinity liquid chromatography as instructed by the manufacturer. PBS buffer was used for equilibration; PBS supplemented with 50 mM imidazole was used for washing the column; while PBS supplemented with 500 mM imidazole and pH adjusted to 7.8 was applied for eluting the protein from the column.

Protein purity was assessed by SDS-PAGE. The expected product molecular weights are 20

52.8, 19.3 and 38.5 kDa for MBP-HFBII, SMBP-HFBII and GFP-HFBII, respectively.

Salts were removed from the eluted fraction using Amicon filters as instructed by the manufacturer and the final protein eluted in distilled water. Protein concentration was measured using the Bradford method and BSA standards and the protein stocks maintained at 4°C for further experiments.

2.4 Results

2.4.1 Design and production of HFBII fusion proteins

Six fusions proteins were designed containing a HFBII domain, aiming to produce hydrophobin-based biosurfactants with an enhanced recovery and additional functionalities. E. coli was used as the heterologous host given its fast growth rate and the wide availability of tools for genetic manipulation. The sequencing results confirm the modified genomic sequence of HFBII, which involved the elimination of introns and optimization of codon usage for its E. coli host (Figure 4).

The SDS-PAGE analysis of the proteins considered in the first design round (yaaD-

HFBII, yaaD-HFBII-CNTbp and yaaD-HFBII-CBD, Figure 5) show a unique predominant band around 14 kDa which is the size of yaaD-HFBII, yaaD-HFBII-CNTbp and lysozyme (Table 6). This similarity makes it difficult to assess the expression level of these two fusion HFBs. In the case of yaaD-HFBII-CBD-, no band is observed at the expected size suggesting a failed expression of this construct.

A ATGCAGTTCT TCGCCGTCGC CCTCTTCGCC ACCAGCGCCC TGGCTgctgt ctgccctacc ggcctcttct ccaaccctct gtgctgtgcc accaacgtcc tcgacctcat tggcgttgac tgcaagaccc gtatgttgaa ttccaatctc tgggcatcct gacattggac gatacagttg acttacacga tgctttacag ctaccatcgc cgtcgacact ggcgccatct tccaggctca ctgtgccagc aagggctcca agcctctttg ctgcgttgct cccgtggtaa gtagtgctcg caatggcaaa gaagtaaaaa gacatttggg cctgggatcg ctaactcttg atatcaaggc cgaccaggct ctcctgtgcc agaaggccat cggcaccttc taa B ATGCAGTTCT TCGCCGTCGC CCTCTTCGCC ACCAGCGCCC TGGCTgctgt ctgccctacc ggcctcttct ccaaccctct gtgctgtgcc accaacgtcc tcgacctcat tggcgttgac tgcaagaccc ctaccatcgc cgtcgacact ggcgccatct tccaggctca ctgtgccagc aagggctcca agcctctttg ctgcgttgct cccgtggccg accaggctct cctgtgccag aaggccatcg gcaccttcta a C ATGCAGTTTT TTGCGGTGGC GCTGTTTGCG ACCAGCGCGC TGGCGgcggt gtgcccgacc ggcctgttta gcaacccgct gtgctgcgcg accaacgtgc tggatctgat tggcgtggat tgcaaaaccc cgaccattgc ggtggatacc ggcgcgattt ttcaggcgca ttgcgcgagc aaaggcagca aaccgctgtg ctgcgtggcg ccggtggcgg atcaggcgct gctgtgccag aaggccatcg gcaccttcta a

Figure 4. HFBII genomic sequence modifications

A: Genomic HFBII DNA from T. reesei (Y11894.1). B: Modified HFBII sequence without introns. C: HFBII exons codon-optimized for expression in E. coli. The SP is signaled by capital letters and the introns with bold format.

Figure 5. Expression of the initial set of HFBII fusion proteins

SDS-PAGE analysis of the expression systems. A: yaaD-HFBII. B: yaaD-HFBII-CNTbp. C: yaaD-HFBII-CBD. All gels contain in lane 1: culture media; 2: cell extract (soluble fraction); 3: cell debris; 4: column flow through; 5: column wash; 6: elution fraction; and the molecular weight markers in the leftmost lane.

Table 6. Protein expected parameters and origin

Size (kDa) IP Origin HFBII (including SP) 8.74 6.97 Synthetic yaaD (N-terminus) 4.20 4.80 Synthetic CBD 17.52 5.07 Synthetic CNTbp 1.43 7.72 Synthetic MBP 40.68 4.81 pPCRWE258 SMBP 10.3 6.65 pPCRSMBP11 GFP 30.32 6.65 pBB082/pNatalia5 yaaD-HFBII 15.27 6.84 pNCM001 yaaD-HFBII-CNTbp 15.64 6.72 pNCM002 yaaD-HFBII-CBD 32.67 5.30 pNCM003 MBP-TEV-HFBII 52.78 4.75 pNatalia6 SMBP-HFBII 19.26 6.72 pNatalia7 GFP-HFBII 39.43 6.59 pNatalia8 Lysozyme 14.3 - Sigma The protein size and estimated isoelectric point were calculated based on the protein sequence using SerialCloner software.

Figure 5 shows the SDS-PAGE analysis of the production and purification fractions of the three initial constructs. As observed in the lane 6 of each gel, no observable protein could be purified from the soluble fraction. Moreover, none of the three overexpression attempts seemed to be successful, as no band of greater intensity was observed where it had been expected from the protein size.

Despite this, a remarkably stable foam was produced from the extract after sonication of yaaD-HFBII expression suggesting that some HFBII-containing product was available.

Therefore, several variations of the purification methods were attempted. The reduction of imidazole concentration in the washing buffer, the incubation of the cell extract at 4°C overnight prior to purification to reduce the oligomerization of hydrophobins57, the use of

Tris buffer instead of phosphate or the addition of SDS to reduce possible protein self- 23

assembly73 and concealing of the His tag were tried. Only the combined use of incubation at low temperatures and Tris buffer (Figure 6), or the addition of SDS at concentrations higher than its CMC (2.3 mg/ml) gave positive results.

Once a feasible purification strategy was found, successive batches of production of yaaD-HFBII were attempted which led us to the confirmation of a silencing effect occurring in our working strains, even when maintained in glycerol at -70°C. This was an unexpected drawback in addition to the low yields obtained. Hence, we designed a second set of HFB fusion proteins to overcome the expression and purification difficulties of the initial set.

Figure 6. Differential purification efficiency of yaaD-HFBII based on buffer selection

SDS-PAGE purification of yaaD-HFBII. Lanes 1-3 show the cell extract, wash and elution fractions of protein in phosphate buffer. Lanes 4-6 show the cell extract, wash and elution fractions of protein in Tris buffer. Initial protein solutions were incubated at 4°C overnight prior to purification. Leftmost lane: molecular weight markers.

The second stage of protein design used a Maltose Binding protein (MBP), a His6 tagged

Green Fluorescent protein (GFP) and a Small Metal Binding protein (SMBP) as N- terminal fusions of the HFBII sequence. The expression plasmids carrying these constructs were pNatalia6, pNatalia7 and pNatalia8, respectively.

Different levels of growth inhibition were observed on the plasmid-carrying strains, both during the construction of the plasmids (Figure 7) and at the protein expression stage

(Figure 8).

Figure 7. Variable transformation efficiencies of HFBII fusion proteins

Panel A shows from left to right the transformation plates from pNatalia6, pNatalia7 and pNatalia8. All tested transformants presumably carrying pNatalia6 were false positives (growing in both ampicillin and tetracycline plates). Two thirds of the strains with pNatalia7 had a correct phenotype (growing in kanamycin but not in tetracycline). All the strains with pNatalia8 showed a correct selection pattern (growing only in kanamycin plates). pNatalia6 was successfully cloned only when glucose was added to the media as a repressor of gene expression (B).

MBP-HFBII, inserted under the lac promoter, was by far the most toxic construct for E. coli. E. coli JM109 had difficulties growing in plates when transformed with pNatalia6.

This strain could only be grown with 30 mM glucose supplemented to the LB media, and low growth temperatures. Even with these conditions the yield was low, making its production impractical.

Figure 8. Effect of temperature and SMBP-HFBII expression on the reduction of total cellular protein E. coli BL21 carrying pNatalia7 for the expression of SMBP-HFBII was grown at 30 and 37°C. For each temperature, the cultures were induced for protein expression. A non-induced control was grown in parallel. The biomass of 1 ml of each growth and expression condition was pelleted and the total protein analyzed using SDS-PAGE. The total protein content can be used as a proxy for bacterial biomass. Lane 1: growth at 37°C no induction. Lane 2: growth at 37°C, induction with IPTG. Lane 3: growth at 30°C induced with IPTG. Lane 4: growth at 30°C, no induction. Note the markedly decreased total protein content observed after SMBP-HFBII induction. Lower growth temperatures seem to palliate this inhibitory effect.

The fusion with SMBP was easily built in a JM109 background but its expression decreased E. coli BL21 biomass when it was grown at standard temperatures (30°C or

37°C, Figure 8). This detrimental effect was managed by lowering the growth temperature to 30°C and induction to 22°C.

Thirdly, GFP-HFBII could be easily built and expressed without compromising bacterial growth at any genetic background or temperature tested. The fusion HFB overexpression was evident in that a dense band on the SDS-PAGE analysis that corresponded with the expected size and fluorescence observed on the cells (Figure 9). Most of GFP-HFBII was produced as inclusion bodies. The addition of 4% glycerol to the LB and the use of M9 media (with 0.5% glycerol and glucose) was tested as it favors the production and solubility of hydrophobic proteins. The result was a highly reduced biomass and overall protein yield (data not shown), suggesting that higher amounts of soluble protein could be harmful for the cells.

Figure 9. SDS-PAGE and fluorescence images of expressed GFP-HFBII.

SDS-PAGE analysis of the total biomass in 1ml of growth culture (A). Lane 1: growth of the GFP producing strain at 37°C and without protein expression induction. Lane 2: growth of the GFP producing strain at 37°C with induction. Lane 3: growth of GFP-HFBII producing strain at 37°C without induction. Lane 4: growth of GFP-HFBII producing strain at 37°C with induction. The cells from the sample in lane 4 (induced GFP-HFBII producing strain) were lysed and the cell debris separated by centrifugation. Lane 5: liquid extract from sample in lane 4. Lane 6: cell debris from sample in lane 4. The fluorescence image of E. coli expressing GFP-HFBII grown on solid media is shown in (B).

Regarding the stability of the expression systems, all proteins were again silenced when the plasmids were kept in the bacteria, regardless of their genetic background. This happened even when they were kept as frozen stocks in glycerol at -70°C.

2.4.2 Purification of SMBP-HFBII and GFP-HFBII by liquid chromatography

SMBP-HFBII and GFP-HFBII could be recovered from the soluble fraction of the cell extract by Ni+ affinity LC without the use of denaturing agents. Both, the N-terminal His tag attached to the GFP fusion partner and the SMBP showed good interaction with the

Ni+ resin and allowed the recovery of the recombinant proteins. The SDS-PAGE analysis

(Figure 10) shows that both bands appear to be lower than expected but in reasonable agreement with their theoretical size. Further verification of protein structure and sequence such as crystallization or mass spectrometry were not carried out. Instead, the functionality of all fusion partners was verified by the existence of fluorescence from

GFP, the Ni+ affinity given by SMBP and the surfactant behavior from HFBII (discussed later).

Figure 10. SDS-PAGE analysis of SMBP-HFBII and GFP-HFBII purifications SMBP-HFBII (left) and GFP-HFBII (right) purification. The eluted (3-6, and 7), washed (2 and 8) and unbound protein (1 and 9) fractions are shown. The bands of interest are signaled by arrowheads. 28

Regarding their purity, the initial eluted fractions of both HFB fusions contain protein contaminants. These could correspond to proteins trapped within GFP-HFBII and SMBP-

HFBII assemblies. The final elution fractions show higher purity leaving only the band of interest and higher molecular weight bands of comparable intensities, which probably are the product of HFB-driven oligomerization or a previous stage of inclusion body formation. The last SMBP-HFBII elution fractions (which had the highest purity) were measured for protein concentration yielding 1.63 mg/L and stored in distilled water at

4°C for further experiments. With these conditions SMBP-HFBII remained in solution and with a stable foaming and self-assembling abilities for 12 months.

2.5 Discussion

The production of biosurfactants has been an area of intense research due to their wide applicability, high performance and biocompatibility3. Unfortunately, the high cost of production and low yields achieved in biological systems, compared to synthetic counterparts, have been a limiting factor for their use. Hence, numerous efforts have been made towards improved production schemes that include the optimization of fermentative conditions19, of downstream recovery processes62,64, the use of cheap and waste substrates8,74,75 and the development of overproducing strains57,59,62. In this work we investigated the potential of a fusion approach to facilitate the recovery of recombinant

HFBs.

Six fusion partners were selected taking into consideration their size, solubility, ease of tracking, reported yields and purification alternatives for an improved HFBII production 29

system. A truncated version of yaaD, a MBP, a SMBP and a GFP were used as N- terminal fusion partners for HFBII, and a CNTbp and a CBD were also tested as C- terminal partners.

The growth behaviors of all strains demonstrated inhibitory effects of soluble HFBII fusions, in agreement with previous reports of HFB expression in heterologous hosts.

Issus including formation of inclusion bodies, low yields and silencing were also observed. Reuter and coworkers (2016) described HFBs, including HFBII, as inducers of inclusion body formation and accumulation for heterologous expressed proteins in plants47. Furthermore, Gutierrez et al. (2013) reported an GFP-HFBI threshold concentration for the formation of inclusion bodies in Nicotiana tabacum and an increased protein accumulation achieved by the addition of p19, a suppressor of post- transcriptional gene silencing58. These suggest that the remarkably high interfacial activity that makes HFBs good candidates for a wide variety of applications might also make them toxic for heterologous hosts, limiting their high-yield production.

Interestingly, the inhibitory effects on cell growth were different for all constructs. The

MBP-HFBII was the most toxic to the point of unviable production, SMBP-HFBII and yaaD-HFBII were moderately toxic and GFP-HFBII was not inhibitory.

The MBP was chosen because of its high solubility and considerably bigger size than

HFBII, which could potentially drive the whole protein to a soluble state. MBP-HFBII construct was under the tac promoter (derived from the trp and lac promoters) which increases the expression efficiency and like lac it is induced by lactose and IPTG and can be repressed by glucose76. Its highly deleterious effect on cell growth could be explained 30

by a leaky expression, suggesting that even trace amounts of protein are harmful for the cell.

The highest protein yield was achieved by GFP-HFBII which is produced in soluble state and inclusion bodies. SMBP-HFBII and yaaD-HFBII yielded a lower amount of protein but seemed to have lower aggregation tendencies. Further experiments varying the media to produce GFP-HFBII showed that the addition of glycerol, which usually increases the solubility of hydrophobic proteins, decreased cell growth and protein production, evidencing a correlation between solubility and toxicity. Furthermore, the several examples of formation of inclusion bodies reported for HFB expression in plants and bacteria could be the hosts’ way to reduce protein availability and subsequent toxicity.

The purification of proteins in inclusion bodies requires a complete denaturation followed by a refolding process. This sequence has been proven difficult to use in some delicate proteins that are unable to completely refold into their active structures without the help of cellular mechanisms such as chaperones. Therefore, the heterologous expression of protein in inclusion bodies is often undesirable, mostly for enzymes and proteins with complex structures. The use of HFB as a fusion tag for other enzymes is then limited by the production of inclusion bodies driven by the HFB domain, so we aimed for the production of soluble HFB fusion proteins. SMBP proved to be a good fusion partner for

HFBII because besides increasing the solubility of the expressed protein, possibly due to a modification of its polarity and hydrophilicity (Figure 11), it also acts as a His6 tag, allowing an easier recovery. The yields obtained with this fusion were lower than those

of GFP-HFBII but practical for our purposes and the protein is stable in aqueous solution for at least 12 months.

All these facts considered suggest that the tradeoff between cell growth (toxicity) and solubility can be balanced with an optimal selection of the fusion partners as well as a careful combination of growth and induction protocols. To overcome this, our results and previous reports emphasize the need for a system-oriented engineering approach for the overproduction of HFB fusion proteins that overcomes their toxic effects on the host.

Possible approaches might be effective secretion systems or a way to modulate its intracellular availability with a synchronic overproduction of hydrophilic/hydrophobic interfaces. Further research on this direction will lead to the design of better production systems for HFBs, HFB fusion proteins and other biosurfactants.

Figure 11. Hydropathy characteristics of SMBP-HFBII.

A shows the hydropathy plot of SMBP-HFBII. Note the two domains with highly differentiated hydrophilicity. B shows a possible SMBP-HFBII structure generated in silico. The charged amino acids are colored in red and blue while the hydrophobic amino acids are shown in grey. The hydropathy plot was prepared using the ProtScale tool from ExPASy31 and the fusion protein sequence.

Lastly, it is worth mentioning that despite the ubiquitous presence of HFBs on filamentous fungi, the lack of genetic tools to modify native hosts or achieve an operative heterologous expression is a hard limit on the applicability of these proteins. Hence, finding a bridge to this biological limit is crucial.

Chapter 3: Taking advantage of a polarity shift: interfacial and self- assembling behaviors of hydrophobin fusion proteins

3.1 Abstract

Native HFBs have a robust and rapid foaming and self-assembling behavior that limits the use of conventional engineering strategies for coating and solution applications. This work examines the self-assembling and interfacial properties of fusion HFBs and the impact of the fusion partner on the HFB surfactant and self-assembling performance.

SMBP and GFP were used to produce fusion proteins containing HFBII, a class II HFB from T. reesei. Our results show that despite the considerable modification done to

HFBII, the fusion proteins behave similarly to the parent HFB. In aqueous solutions,

SMBP-HFBII and GFP-HFBII maintain a surfactant behavior, with an estimated CMC <

5 mg/l. SMBP-HFBII is also prone to form nanobubbles (50-60 nm) and thin films at liquid-air interfaces. It is further demonstrated that SMBP enhances the affinity of HFBII for polar substrates. SMBP-HFBII coatings formed on hydrophilic substrates (mica and glass) generate homogeneous protein distributions that reverse surface wettability increasing the water contact angle (WCA) 460% and 70%, respectively, due to the enhanced dual-domain amphipathic character. This work shows that fusion hydrophobins can be greatly promising to expand the application potentials of its native parent proteins.

3.2 Introduction

HFBs’ spontaneous self-assembly induces the formation of oligomers and interfacial structures of interest for their biological roles and technical applications. Some of these applications include the production of protein sensors or smart materials that rely on a uniform protein distribution on the solid surface. Unfortunately, the use of standard methods that do not account for HFB unique characteristics often render uncontrolled foaming and heterogeneous results. Therefore, understanding the underlying mechanisms of HFB self-assembly and developing ways to use it in a controlled manner is required for an operative use of their properties. In this chapter two fusion HFBs are used to investigate the impact of the fusion partner on the native HFB surfactant, self-assembling and immobilization behaviors.

HFBII’s properties have been extensively studied due to its role on the gushing effect§ of carbonated beverages77,78. Light scattering experiments have shown that HFBII forms stable tetramers in solution at concentrations ranging from 0.5 to 10 mg/ml, in a wide range of pH (4 - 7) and temperatures (6 – 60 °C). Hydrophobic interactions drive oligomerization processes that conceal the hydrophobic patches of each molecule from the aqueous environment48,57,73,79,80. However, the importance of other interactions involving charged side residues have also been reported60,81 giving a clearer picture of the protein behavior in solution.

§ Typically observed in carbonated beverages, it is the spontaneous formation of high amounts of foam and gas that is freed from the solution, increasing the pressure inside a closed container and in some cases causing its abrupt opening. 35

HFBII is also known to form organized monolayers and elastic membranes28,82–84 at the air-water interface. It also has been reported to achieve a remarkable decrease in surface tension at protein concentrations as low as 20 mg/l85, thus enhancing the stability of foams37, microscopic “needle-like”79 structures and nanobubbles77,78. Its exceptional foaming ability is highly valued in the food and cosmetics industries but it is also inconvenient for the brewing and other bioprocess industries in which excessive foaming or assembling is unwanted86,87.

Additionally, previous studies suggest that HFBII and HFBII fusion proteins rapidly bind to polar and non-polar solids on a variable manner depending on the surface chemistry and deposition method66. Generally, they are weakly bound and easily washed with water85, buffer or 0.5 - 2% SDS solution88. HFBII binding to hydrophilic materials has produced imperfect films and other variable morphologies described as “pimples”,

“voids”, “trilayers” or layers with high degree of mosaicity28,32,55,89. Similar structures formed by HFBI, another class II HFB from T. reesei, correlate with the variable macroscopic effects on the surface properties after immobilization such as a variable layer thickness and wettability53. The heterogenous results achieved using HFBII, HFBI and other native HFBs have limited their use as protein anchors and surface modifiers.

This work explores the effects of a fusion strategy on the surfactant and self-assembling behaviors of HFBII. We aim to modulate the HFB native characteristics by means of a fusion partner. Our results show that HFBII fusions maintain their surfactant and self- assembling behavior. However, using SMBP, a highly hydrophilic metalloprotein, produces a shift in the overall protein’s polarity that increases its affinity for hydrophilic

substrates. Uniform protein coatings on such substrates were achieved using SMBP-

HFBII fusion. It is demonstrated that the fusion strategy is a powerful avenue to modulate

HFB behavior to better fit a broader range of applications.

3.3 Materials and Methods

3.3.1 Materials

Mica (Electron Microscopy Sciences, Hatfield, PA, USA), glass (Cida, Leawood, KS,

USA) and polyvinyl chloride (PVC) slides (Benz Microscope Optics Center Inc, Ann

Arbor, MI, USA) were used as solid substrates for protein immobilization. SMBP-HFBII,

GFP and GFP-HFBII were produced as described in chapter 2. NaOH, HCl and glutaraldehyde were bought from Fisher Scientific (Pittsburgh, PA, USA).

Atomic force microscopy experiments were carried out with a Keysight 5500

Environmental Scanning Probe Microscope (Santa Rosa, CA, USA) using silicon cantilevers with tip radius < 10 nm and nominal spring constant of 42 N/m (Nano World

Innovative Technologies, Neuchâtel, Switzerland) and 2 N/m (Mikromasch, Watsonville,

CA, USA). XPS measurements were performed on a PHI Versa Probe III XPS system

ULVAC-PHI (Physical Electronics Inc., Chanhassen, MN, USA). Water contact angle was measured with an optical contact angle meter (Kyowa Interface Science Co, Japan).

3.3.1 Assessment of surfactant behavior by light and fluorescence microscopy

To test the interfacial behavior of the fusion proteins, 100 μl of 0.1 mg/ml solutions of

GFP and GFP-HFBII were sonicated for 10 min and mixed in an orbital shaker at 1400

rpm for 30 min without temperature control. Then, 5 μl of the protein solution were deposited between glass microscope slides and observed with a confocal microscope on fluorescence and bright field modes. Images were taken at the University of Minnesota

Imaging Centers.

3.3.2 Protein deposition on solid substrates

To identify the self-assembled structures formed by SMBP-HFBII, we used three different protein deposition methods on mica slides. The immobilized protein structures were then examined with AFM. Each method differed in the interactions between the liquid-solid or air-liquid-solid interfaces. For clarity a graphic scheme depicting all methods is shown in Figure 12.

3.3.2.1 Transfer of protein structures by direct contact with the air-water interface

Droplets of SMBP-HFBII solutions (250 μl) with concentrations ranging from 10 ng/ml to 5 μg/ml prepared by serial dilutions from a 5 μg/ml stock were placed over a Parafilm sheet. The droplets were incubated at 4°C and 100% relative humidity overnight. The droplet surface was gently touched with freshly cleaved mica slides. Then, the slides were preserved as such or washed by immersion in a water bath prior to drying at room temperature and further analysis. In this process the solid surface touches the air-liquid interface once. This method is modified from Asakawa et al. (2009)66.

3.3.2.2 Immersion of solid substrates in a protein solution

The solid substrate was immersed in SMBP-HFBII solutions of 50, 5 or 0.5 μg/ml in distilled water and incubated overnight at 4°C. Then, the substrate was recovered and

thoroughly washed by immersion in a water bath. The excess water was dried slightly touching with a Kimwipe on the corner of the solid and maintained at room temperature in sterile conditions. With this method the solid substrates cross the air-liquid interface twice, at immersion and recovery.

3.3.2.3 Free adsorption of SMBP-HFBII from aqueous solution

Cleaned substrates were placed in a sample holder inside a reactor (Figures 12C and

12D). The reactor was filled with 45 ml of sterile water pH 5 (adjusted with diluted HCl).

Then, 450 μl of a 50 μg/ml SMBP-HFBII solution were gently added to the water surface, avoiding any contact with the solid, for a final concentration of 0.5 μg/ml. The samples were maintained in the reactor, overnight, at room temperature and with mild stirring. Then, they were washed by a continuous stream of distilled water (50 ml/min) for 10 min to wash off unbound protein. The coated samples were then collected and dried at room temperature in sterile conditions. In this process the solid never encounters the air-liquid interface. For AFM experiments the protein coatings were fixed by immersion in a 5% (v/v) glutaraldehyde solution for 30 min, then they were thoroughly washed in distilled water and dried at room temperature.

Figure 12. Methods used for protein deposition on solid substrates A: Immersion of solid substrates in the protein solution; B: Transfer of protein structures by direct contact with the air-water interface; and C: protein adsorption from the liquid phase. D: experimental setup for the protein adsorption method showing the adsorption reactor (left) and the washing set up (right).

3.3.3 Analysis of protein interfacial assemblies by AFM

Mica slides coated with the different methods described above were analyzed with AFM to visualize the different protein structures. Image sizes ranged from 1 to 50 μm2, and the 40

typical scan rate was 0.75 lines/second for a resolution of 512 × 512 pixels. The images were taken at room temperature and low humidity (nitrogen flushed atmosphere) in tapping mode with the stiffest cantilever. Slides with adsorbed protein were scanned with the most flexible cantilever in contact mode.

The data was visualized and prepared using Gwyddion software. Image preparation involved flattening, elimination of scratches using the 2D FTT tool and coloring adjustments. Grains (agglomerates) were marked by height and curvature threshold and their size distribution estimated with the maximum inscribed disc radius.

3.3.4 Analysis of surface chemical composition by XPS

XPS measurements were performed using a monochromated Al Kα X-ray source (1486.6 eV). The base pressure was 5.0x10-8 Pa. During data collection, the pressure was 2.0x10-6

Pa. All samples were mounted on a piece of double-sided adhesive tape on a sample holder. The samples were non-conductive and charge neutralization was used. Two or three spots were analyzed on each sample. A 200 µm X-ray beam with a power of 50 W or a 100 µm X-ray beam with a power of 100 W was used to scan across a sample area of

1400 × 500 µm. The survey spectra were collected using 280 eV pass energy and 1.0 eV/step. The atomic percentages were calculated from the survey spectra using the

Multipak software provided with the XPS system.

3.3.5 Surface wettability measurements

Wettability was tested with the sessile drop method using a 30 μm diameter capillary and distilled water. For each surface, the values reported are the average of 5 droplets (right

and left angle) ± standard deviation. The contact angle was calculated through the tangent method. FAMAS software was used for image analysis and angle calculation. Special care was taken to avoid any confounding effects of residual salts or other contaminants on the surface, so coated samples and controls were thoroughly washed with distilled water and dried at room temperature before measurements.

3.4 Results

3.4.1 Interfacial activity and foaming effect of HFBII fusion proteins

The surfactant behaviors of GFP-HFBII, SMBP-HFBII and GFP in solution at a concentration of 0.1 mg/ml were assessed by monitoring their foaming ability. The bright field images of the foams formed by each protein solution show increased foaming in the

SMBP-HFBII and GFP-HFBII samples compared to GFP (Figure 13, panels A-C).

Both fusion HFBs stabilize bubbles of a wide range of sizes (2 μm to mm scale).

Flocculation is observed in both solutions, enabling the formation of intricate structures that resemble dendrite-like organizations (figure 14A and 14B). However, coalescence and formation of bigger size bubbles** is only evident with the GFP-HFBII fusion. In general, SMBP-HFBII stabilizes foams of smaller and more uniform size. The formation of stable foams led us to infer that the CMC of both fusion proteins is below 0.1 mg/ml.

** Flocculation in this context refers to the process of contact and adhesion whereby the particles dispersed in solution that forms larger-size clusters, the total number of particles remains approximately constant. Different than coalescence where the flocculated particles mix together increasing their size while decreasing the number of total particles2. 42

Figure 13. Interfacial and foaming activities of GFP-HFBII and SMBP-HFBII. Solutions of SMBP-HFBII (A) and GFP-HFBII (B, D) stabilize micrometer-size air bubbles, showing an increased foaming activity when compared to GFP (C). The fluorescence images display a differential solution behavior of GFP-HFBII (E) and GFP (F). The differences observed between E and F are attributed to the HFBII moiety. Arrowheads point to gas-liquid interfaces.

Like HFBII, the action of shear forces (vortexing or vigorous shaking) on GFP-HFBII solutions generates needle-like and channel structures (figure 14C and 14D).

Interestingly, these structures are remarkably resistant and maintain their structural integrity for days after drying.

Additionally, the fluorescence images (figure 13E and 13F) show a differential GFP solution behavior with and without the HFBII domain. The C-terminal HFBII seems to increase the protein presence at the air-water interface and inhibits the formation of large protein deposits, possibly formed during the harsh sonication and high temperature treatment. 43

Figure 14. Microstructures formed by self-assembling of GFP-HFBII stabilized foams Bright field images of a droplet deposited on a glass slide from a GFP-HFBII protein solution at 0.5 mg/ml concentration. Images A and B come from solutions that were not subjected to shear forces (vortexing). Note the long, intricate, dendrite-like structures formed. Images C and D come from solutions that were vortexed before transfer to the glass slide. Note the long and seemingly organized channels formed. Shear forces determine the type of microstructure formed by the GFP-HFBII stabilized foams.

3.4.2 Protein self-assembled micro and nanostructures

The self-assembling capabilities of SMBP-HFBII and the effect of the HFB fusion partner were investigated. Mica slides with SMBP-HFBII structures transferred by direct

contact between the solid and the air-water interface were scanned using AFM. The mica surfaces showed three types of protein structures: micro and nano-spheres (presumably stabilized air bubbles), patterned films and smooth and tightly packed films.

Micro and nanobubbles were the predominant structure at concentrations above 5 µg/ml.

We could observe a positive correlation between the protein concentration and the number of bubbles on the mica surface. Below 5 μg/ml nanobubbles were rare and easily washed off but at higher concentrations (5 - 100 μg/ml) they were abundant and prone to agglomerate forming bigger size assemblies up to the micrometer range. A grain size analysis of these AFM images (Figure 15) shows a predominant sphere radius of 50 - 60 nm followed by a smaller peak at ~100 nm (10 fold less frequent) and fewer examples of bigger agglomerate sizes that go up to ~ 1 μm.

Figure 15. AFM images of SMBP-HFBII globular assemblies. Dendrite like organizations formed by globular assemblies (A) were found when the self-assembled structures in a 5 μg/ml SMBP-HFBII solution were transferred to a mica slide by contact with the air-water interphase. The inscribed disc radii (R) distribution of the globular assemblies found in A is shown in (B). Note the predominant radius size around 50 nm.

At protein concentrations below 5 μg/ml two different film structures were revealed.

First, we could observe a patterned film with a height difference between peaks and valleys within 3 nm. This film showed a wide coverage of the surface and highly resembles previously described structures formed by HFBII in Langmuir-Blodgett coatings55. Second, a smooth, flat and a tightly packed film (Figure 16) was observed when working at concentrations in the order of ~ 10 ng/ml. This smooth film had a limited coverage of the surface and seemed to grow by the progressive junction of several smaller islands with the same morphology (figure 16C).

The three abovementioned structures (nano and microbubbles, patterned films and smooth films) coexist (Figure 16D) in samples that were not subjected to washing after the protein deposition from diluted solutions.

Figure 16. Topography of SMBP-HFBII self-assembled film structures AFM topography images. The protein structures were transferred by contact with the air-liquid interface from SMBP-HFBII solutions with various concentrations. A shows the predominant topographies transferred from a 0.1 μg/ml protein solution (detail scale 0.5 μm). B and C correspond to a 0.01 μg/ml solution. D shows the relative position of protein agglomerates and films.

3.4.3 Immobilized SMBP-HFBII at the solid-liquid interface

Using HFBII and HFBII fusion proteins for surface functionalization and protein immobilization requires a uniform protein distribution. This is usually hampered by the

HFB characteristic rapid and uncontrolled self-assembling. Therefore, we investigated the effect of SMBP as a fusion partner for HFBII on the protein distribution of coatings made on mica using XPS and AFM.

Mica is composed mainly by O and Si atoms with trace amounts of Al and other elements. Mica does not contain nitrogen (N). Therefore, the presence of N atoms on its surface with a characteristic XPS binding energy of 400 eV can be used as a reporter of protein content. Moreover, a peak at 288 eV on the XPS spectra indicates the existence of amide groups, also related to protein structures90. Thus, the relative protein content of different samples can be inferred comparing the N% among samples and the surface coverage comparing the respective N% to Al% or N% to Si% ratios. We used these peaks on the XPS spectra to confirm the protein nature of the coatings and their relative elemental composition (Figure 17 and Table 7).

The protein deposited by immersion of the mica slide in the protein solution had an uneven distribution and a patterned structure. When the protein was freely adsorbed on the surface (without touching or passing through the air-water interface and without mechanical forces involved) the protein coating had a homogeneous topography. The protein adsorption limited the appearance of nanobubbles on the coating and favored a fabric-like distribution of protein on the surface (figure 17A). This structure spans throughout the whole scanned area showing a uniform and even topography with a height range within the nm. It was not possible to further resolve the ultrastructure of this film or measure its depth with AFM techniques.

Figure 17. Surface topography and protein content of SMBP-HFBII coatings analyzed by AFM and XPS Mica slides were coated by adsorption (A) or immersion (B) of the substrate from a 0.5 μg/ml SMBP-HFBII solution. A higher resolution detail is shown in A with a scale bar of 0.2 μm. The C1s and N1s high resolution spectra of each sample show binding energy peaks at 400 eV and 288 eV, confirming the protein nature of both coatings.

The XPS C1s and N1s high resolution spectra of both coatings confirmed their protein nature with a marked peak at 288 eV and 400 eV. Furthermore, comparing the performance of both coating methods, their relative elemental composition (Table 7) shows a higher protein content and a larger surface coverage achieved by the protein adsorption.

Table 7. Surface relative atomic composition of SMBP-HFBII coated mica Deposition method N (%) C (%) O (%) Si (%) Al (%) N% : Al% N% : Si% Protein adsorption 0.5 μg/ml Spot 1 5.6 39.0 39.4 7.6 6.4 0.88 0.74 Immersion 0.5 μg/ml Spot 1 1.5 40.8 46.7 5.2 3.7 0.41 0.29 Spot 2 1.8 23.5 54.1 9.6 7.1 0.25 0.19 Protein adsorption 30 ng/ml Spot 1 1.5 26.3 52.2 8.3 9.1 0.16 0.18 Spot 2 1.7 23.9 52.8 8.9 9.9 0.17 0.19 Mica crystals were coated by protein adsorption from a SMBP-HFBII solution with 0.5 μg/ml (a) and 30 ng/ml (c) protein concentrations. For comparison, coating by immersion from a SMBP-HFBII solution with 0.5 μg/ml (b) was also tested.

3.4.4 Effect of SMBP-HFBII coatings on material properties

We investigated their macroscopic effects on the wettability and surface composition of

SMBP-HFBII coatings on different materials. The materials chosen had different chemical properties to assess the behavior of SMBP-HFBII for a variety of possible supports. They were mica (highly hydrophilic), glass (moderately hydrophilic) and PVC

(hydrophobic). Distilled water and BSA solutions were used as controls.

The surface wettability of hydrophilic substrates (mica and glass) became considerably higher when immersed in water (Table 8). This effect is due to the dissociation of SiOH groups on their surface, which results in a negative charge (SiO-) and increased hydrophilicity91. The WCA of glass decreased a 55% and mica’s WCA was below the detection limit. This effect is not observed on PVC. Protein (BSA or SMBP-HFBII) in solution modified the WCA of all substrates in a different manner.

Table 8. Water contact angle of protein-coated materials Treatmenta Mica Glass PVC Untreated 9.6 ±0.8 38.8 ±1.4 73.7 ±1.2 Water control BDLb 17.0 ±1.8 72.1 ±1.7 SMBP-HFBII 54.3 ±4.5 61.2 ±1.8 64.1 ±4.0 BSA 23.0 ±3.5 25.2 ±3.4 70.2 ±3.6 SMBP-HFBII (immersed)c 25.5 ±3.2 44.2 ±5.7 80.4 ±2.1 SMBP-HFBII (30 ng/ml) 11.4 ±1.1 27.7 ±3.7 72.2 ±9.1 a Protein was adsorbed from a 0.5 μg/ml solution unless otherwise noted. b Below detection limit c For comparison with the adsorption coating method, this sample was immersed in the protein solution as described in materials and methods.

Adsorbed SMBP-HFBII at 0.5 µg/ml achieved reversed the wettability of all substrates. It increased hydrophobicity of mica and glass and slightly decreased wettability for PVC

(Figure 18). Their WCAs were 54° ± 4.5°, 61.2° ± 1.8° and 64.1° ± 4.0°, respectively, denotating a 460% and 70% higher and 13% lower WCA when compared to the untreated material.

Substrates coated by immersion did not achieve the same results. Although mica and glass became more hydrophobic, their WCA increase was not as pronounced as the one achieved by protein adsorption (only 165% and 14% respectively). PVC’s WCA was uneven throughout the surface suggesting heterogeneous coating results.

Figure 18. Modulation of materials’ surface wettability by SMBP-HFBII coatings Mica, glass and PVC surfaces coated by adsorption of SMBP-HFBII from a solution with 0.5 μg/ml of protein shows a marked increase in the hydrophobicity of mica and glass and a discrete reduction on the surface hydrophobicity of PVC. These changes are demonstrated by the increase and reduction of the WCA for each material.

XPS experiments were carried out to assess the surface composition of adsorbed protein coatings on mica, glass and PVC. None of the substrates used contains N, therefore, N can be used as a reporter for protein content on the surface. However, given their different elemental compositions, it is not possible to compare their surface coverage using N ratios. The N1s high resolution spectra of all coated materials show peaks at 400 eV, confirming the presence of protein. Additionally, the N% of mica and glass are approximately the same (5.6% and 5.3%) and slightly higher than the N% of protein coatings on PVC (3.8%, Figure 19).

Figure 19. Surface protein content of different materials coated with SMBP-HFBII analyzed by XPS The N1s high resolution spectra of mica, glass and PVC (A) show a marked peak at 400 eV, demonstrating the presence of amide groups related to protein on all samples. The relative atomic composition of each surface (B) shows a higher nitrogen content (N%) found on mica and glass, the most hydrophilic substrates.

3.5 Discussion

In this work we investigated the surfactant and self-assembling behaviors of two HFBII fusion proteins, aiming to analyze the effect of the fusion partner on the native HFB properties.

Regarding their solution and surfactant behaviors, GFP-HFBII and SMBP-HFBII displayed the characteristic foaming of the native HFBII at 0.1 mg/ml (0.01% w/w), suggesting a considerable decrease of surface tension. This concentration can be used as an upper limit for their CMC value. Thus, we estimated that their CMC is more than 10 times lower than the CMC of SDS (1.7 - 2.3 mg/ml), making both HFB fusions remarkably better surfactants than the synthetic detergent. 53

Additionally, each fusion HFB showed differential surfactant behavior, demonstrating the influence of the fusion partner on the overall protein properties. GFP-HFBII favored the coalescence of foams while SMBP-HFBII maintained stable bubbles of smaller diameter.

This effect seems to be due to a steric hindrance of the much bigger GFP moiety on the

HFBII natural assembling. SMBP has a comparable size to HFBII and did not show coalescence as much as GFP-HFBII.

An interesting consequence of the higher coalescence of GFP-HFBII foams was that when subjected to shear forces, the stabilized air bubbles solidified in regular mm-long channels. Without shear forces, the microbubbles self-organized in dendritic patterns upon dehydration. These structures showed a remarkable stability being able to resist a complete drying process and remained intact on the glass surface.

GFP-HFBII showed lower precipitation after prolonged sonication compared to GFP.

This observation suggests a higher endurance to harsh conditions of the fusion protein given by the HFBII domain. Such result is encouraging towards the use of HFBII fusion proteins for enzyme stabilization, or to increase enzyme compatibility with hydrophobic media in multiphase systems such as hydrophobic paintings92 or foams.

We also studied the self-assembling of SMBP-HFBII to assess the effect of our fusion strategy on HFBII’s natural organization. The establishment of nanobubbles and thin films like those formed by HFBII demonstrated that the HFB self-assembling capabilities remained active despite the considerable modification performed on the native protein.

The endurance of the protein stabilized structures was remarkable. Even after sonication, transfer to the mica crystal and drying, those structures were intact on the surface and could be imaged with AFM for weeks after deposition. Moreover, they could stand drastic changes in relative humidity as they were scanned under N2-flushed atmosphere and low relative humidity or ambient environments. It is also worth noticing that the existence of such high number of globular structures at ~ 5 µg/ml pushed down our previously estimated limit for SMBP-HFBII’s CMC. This new estimated value (~ 5

µg/ml) demonstrates a better surfactant activity of the HFB fusion than surfactin, a highly active lipopeptide from B. subtilis7,74.

Additionally, as would be expected for a HFB derived protein, the deposition method on mica and resulting interaction of protein in different interfaces (solid/liquid/gas) triggered the formation of different protein structures. When the mica crossed the air-water boundary, uneven films were created with attached globular assemblies and “patterned” segments. We could also observe areas with a smooth and tightly packed layer. To our knowledge it is the first time that such a uniform structure has been imaged on HFBII, any HFBII fusion protein or other hydrophobin. This smooth film was repeatedly observed at extremely low protein concentrations and seemed to grow by the progressive junction of smaller islands during the sample drying (figures 16B and 16C). This observation together with its topography leads us to infer that it might correspond to the protein monolayer described for HFBII at the air-water interface. Unfortunately, this type of film could not be reproduced along extended areas of the solid surface or with higher protein concentrations.

The distribution of SMBP-HFBII in a 3-phase environment (solution/solid/air) could be inferred from the relative localization of all the above-mentioned structures. At low enough concentrations (< 5 μg/ml), the micro/nanobubble count decreases considerably and reveals underlying protein films. The patterned film seems to be formed by the interaction and reorganization of nanobubbles. The smooth film was formed at the air- water interface and could be observed only at extremely low protein concentrations and long incubation and drying periods. These suggest a dynamic relationship and a hierarchical organization of SMBP-HFBII self-assembled structures that depends on the existence of bubbles in solution and the proximity to the solution-air interphase (Figure

20). Hence, the rapid decrease in surface tension achieved by HFBII is probably due to a fast migration of stabilized air bubbles towards the interface, instead of simple protein diffusion.

Figure 20. Hypothetical HFBII-fusion protein distribution in the proximity of the air-liquid interface Smooth and tightly packed protein layers (i), more disorganized patterned films (ii) and micro and nanobubbles (iii) coexist and interact among each other.

Next, we investigated the effects of our SMBP fusion on the properties of immobilized

HFBII on solid surfaces. We could observe how the deposition method, accordingly, the structures on the material’s surface, played a crucial role on the resulting protein distribution and surface modifications. Immersion of the sample in the protein solution

(thus, using mechanical forces to go through the air-water interface) achieved films that resemble the Langmuir-Blot coatings described by Kisko et al.55 and the Langmuir

Through coatings described by Stanimirova et al.28 for HFBII. The surface of coated mica, glass and PVC had uneven N% and WCA. On the other hand, the adsorption of

SMBP-HFBII generated homogeneous fabric-like coatings that were highly resistant to abrasion by the cantilever tip. Such fabric structure might be formed by the adsorption of protein in solution, i.e. in oligomeric form. We could infer from the observed data that common coating techniques such as Langmuir-Blodgett or Langmuir-through are not ideal for HFBs, due to their rapid self-assembly and differential structure formation at interfaces and bulk solution.

Regarding the effect of SMBP on the fusion protein, the higher N% found on mica and glass suggested an increased affinity and stability of SMBP-HFBII on polar surfaces.

This demonstrated that modifying the native HFBII with a highly hydrophilic metalloprotein greatly improved the behavior previously described for HFBII66,88,93.

SMBP not only increased HFBII solubility but also its interaction with hydrophilic substrates achieving noticeable surface wettability modifications at protein concentrations in the order of 0.5 μg/ml.

Our results showed that the interfacial and self-assembling properties of GFP-HFBII and

SMBP-HFBII display the robust processes driven by the HFBII moiety. We also demonstrated that the overall protein behavior is modulated by the fusion partner. These results are encouraging towards the rational enhancement of the HFBs’ properties by a fusion strategy. A careful selection of fusion partners can facilitate HFB purification and enhance protein polarity, solubility and interaction with hydrophilic substrates as was the case of SMBP. These fusions, in turn, could be further modified for the construction of modular surfactant proteins to be used in a wider array of technical applications.

Chapter 4: Biocontainment of fungal spores by SMBP-HFBII coatings

4.1 Abstract

Fungi play important roles in plant and animal disease. They are important factors for food loss and waste throughout the supply chain. Spores are the vehicle for fungal dispersion and for attachment to healthy products before infection. HFBs have been reported to coat fungal spores and hyphae, being essential for their hydrophobicity and interaction with plant surfaces. Here we explore the effect of a HFB fusion protein on glass affinity for fungal spores and subsequent spore adhesion. Glass slides coated with

SMBP-HFBII, an engineered fusion HFB, showed increased adhesion of B. cinerea spores, but this result was dependent on pH and spore concentration. The highest spore adhesion was found at pH 9, condition at which the high dispersion observed on the results limited our ability to conclude significant differences between the coated and uncoated materials. The most significant increase achieved by the protein coating was found at pH 5. It was further observed that spore concentrations above ~106 spores/ml promoted clumping and prevented spore adhesion to surfaces.

4.2 Introduction

Hydrophobins are an interesting group of proteins from a biological and technical perspective due to their self-assembling properties, interfacial activity and important roles on microbial physiology and pathogenicity29,1,94. In this chapter we explore the effect of

SMBP-HFBII, an engineered fusion HFB with high affinity for polar substrates, on the adhesion of Botrytis cinerea spores to glass. We aim to shed light on the spore-surface interactions leading to spore adhesion to synthetic surfaces and to explore the use of

HFB-functionalized materials towards biocontainment purposes.

Fungal pathogens play important roles in crop and food losses. High value fresh products such as flowers95, fruits and vegetables are especially vulnerable to decay and waste96.

Current efforts to control fungal diseases rely in the disease prevention at the crop stage, resistance breeding of specific cultivars and controlled atmosphere environments during transport and storage97. Unfortunately, these efforts have been insufficient to eliminate fungal diseases and oftentimes impose selective pressures that increase the resilience of these organisms.

Several characteristics of the life cycle of fungal pathogens make them very difficult to eliminate once they have been introduced in a susceptible population. For instance, B. cinerea is an excellent example of a versatile and resilient infectious agent94,98. It has a broad host range (over 200 species) that acts as reservoir and facilitates post-harvest cross-contamination. It also has several mechanisms of propagation such as direct contact with infected hosts or through animal and environmental vectors (birds, wind, rain droplets) carrying spores and other propagules99. Additionally, it initiates infection rapidly after sensing conductive environmental factors such as temperature and humidity fluctuations, which are extremely difficult to control in the field98,100. Furthermore, B. cinerea’s metabolic flexibility favors its rapid development of resistance to fungicides101–

105.

Specialized survival structures (i.e. sclerotia) withstand harsh environmental conditions and rapidly reinitiate growth when circumstances are conductive, producing large amounts of dispersal structures (spores). These quiescent structures (sclerotia and spores) remain unnoticed in seemingly healthy products which are then harvested, transported and stored along with susceptible hosts, increasing the potential of post-harvest transmission and product loss94.

Therefore, alternative approaches to reduce fungal diseases are desirable. Recent efforts have been made to improve early detection methods such as recognition of asymptomatic carriers106, and to design methods to monitor the spore load on the environment107. At the post-harvest stage, the use of edible coatings that increase the shelf-life of fresh products has also been widely used108.

In this work we focus on an early step of disease development, the adhesion of fungal spores to solid surfaces. Our results evidence a positive correlation between materials’ hydrophobicity and spores’ adhesion. It was also observed that SMBP-HFBII coatings that do not increase materials hydrophobicity significantly, increase spore’s adhesion to glass, but this effect depends on pH and spore concentration. Further research is required to optimize the biocontainment features of HFB coatings.

4.3 Materials and methods

4.3.1 Materials and microbial strains

Buffer salts and acids were bought from Fisher Scientific (Pittsburgh, PA, USA). Potato dextrose agar (PDA) was bought from Difco. Glass slides (Cida, Leawood, KS, USA) and polystyrene from petri plates were used as solid substrates for protein deposition and spore adhesion tests. Glass slides were treated with a piranha wash to increase their hydrophilicity. Spores from B. cinerea strain BO5.10 were used for adhesion tests.

Microscopy was carried out at the University of Minnesota Imaging Centers with a Nikon

A1 Spectral Confocal Microscope.

4.3.2 Preparation of solids substrates

Glass slides were used as provided by the manufacturer or coated by adsorption of

SMBP-HFBII from a 30 ng/ml solution using the method and equipment described in

Chapter 3. To test the possible coating leaking some of the coated slides were also subjected to protein crosslinking.

4.3.3 Botrytis cinerea growth conditions

B. cinerea strain BO5.10 was stored as frozen spore stocks in 50% glycerol, with a spore concentration of ~106 spores/ml. Fresh cultures were grown from the frozen stock in

PDA plates and maintained in darkness and at room temperature for 10 - 15 days. At this time point the fungi had sporulated.

4.3.4 Air dispersion of spores

An initial test of the effect of hydrophobicity on spore adhesion was assessed by air- dispersed spores. The sporulated culture (petri dish) was set at the bottom of a closed chamber and the testing materials were fixed on the top of the chamber. A stream of air was directed to the spores, which were carried out and dispersed by the air flowing through. After 30 min of continuous flow, the air stream was stopped, and the test substrates collected. The substrates were then visualized with light microscopy. Images of 5 randomly positioned fields on the surface were captured. Each image was inspected, and the number of spores manually counted.

4.3.5 Adhesion of spores in solution

Spore adhesion tests in liquid environments were developed based on Doss et al. 1993109.

Spores were collected by washing the surface of the fungal mat with distilled water of pH

5, 6.2, 7.5 and 9. The pH was adjusted with HCl or NaOH. The spore suspension was filtered with a cheesecloth to remove hyphae and bigger structures. The concentration of spores was determined with a hemocytometer and adjusted to ~1x106 spores/ml for all experiments, unless otherwise stated. Adhesion tests were done by placing 1 μl of the spore suspension on top of the solid substrates and incubating for 30 min at room temperature. Then, the substrates were washed in a water bath twice, dried at room temperature and observed with light microscopy.

4.3.6 Quantification of spore adhesion by light microscopy

The samples prepared as described above were observed using bright field mode. Images were captured from 9 adjacent fields centered on the point of droplet deposition for each 63

sample. Each image was inspected, and the number of spores manually counted. The average of the 9 frames inspected were used to describe each sample. The results reported are the average and standard deviation of three samples per treatment.

4.4 Results

4.4.1 Effect of surface hydrophobicity on spore adhesion to substrates

As shown in Figure 21, polystyrene slides (the most hydrophobic material tested) could retain twice the number of spores compared to the untreated glass (medium hydrophobicity) and ten times more spores than the piranha-washed glass (the most hydrophilic substrate). The hydrophobicity of the materials tested played a significant role on spore adhesion.

Figure 21. Effect of material hydrophobicity on B. cinerea spore adhesion to surfaces The adhesion of air-dispersed spores to different materials was tested (A, bars). The hydrophobicity of the materials was estimated by measuring the WCA (A, line) of all surfaces. B shows the experimental set-up used for the spore dispersion.

4.4.2 Effect of pH and protein coating on the adhesion of spores to glass substrates

The effects of SMBP-HFBII and pH on spore adhesion to solid substrates were tested using spore solutions of different pH dispersed over coated and uncoated glass slides.

As observed in Figure 22, solutions of pH 9 considerably favor the adhesion of spores to all substrates. Slightly acidic or basic solutions (pH 5 and 7.5) had a lower and similar adhesion results. The lowest level of spore adhesion was observed at pH 6.2, which is the estimated isoelectric point of SMBP-HFBII. The same pH dependency was observed for all materials tested (coated and uncoated), suggesting the pH primarily impacted the spore wall properties.

For all pH levels, the coated samples had a slight increase in spore adhesion to the substrate, mostly when the protein coating was crosslinked. However, given the high dispersion of our results compared to the differences between treatments, the protein coatings only showed significant increase in spore adhesion at pH 5 and 7.5. At pH 5,

SMBP-HFBII crosslinked coating increased the adhesion of spores to the solid substrate a

130%. Overall, the effect of the protein coating appeared to be much lower than the effect of pH.

Figure 22. Effect of pH and SMBP-HFBII coating on B. cinerea spore adhesion. Spore solutions of pH 5, 6.2, 7.5 and 9 were tested for adhesion to SMBP-HFBII coated and uncoated glass slides. For comparison, samples with a fixed protein coating were also tested. The bars represent the mean of 3 samples and the error bars the standard deviation. Different letters mark statistical difference within a pH level with a 95% confidence. Note the much larger effect of pH compared to the effect of type of surface.

4.4.3 Effect of spore concentration on their adhesion behavior

To estimate the limits of a biocontainment approach, we investigated the effect of spore concentration on their adhesion to different substrates. Adhesion tests were carried out with spore solutions ranging from 4 ×105 to 5 ×107 spores/ml at pH 7.5. We could observe that above ~107 spores/ml, the spores started to show clumping and formation of heavier agglomerates, presumably due to intensified spore-spore interactions when adhesion got accumulated. These agglomerates were easily washed away from the surface resulting in an uneven distribution and large dispersion of results (Figure 23 A

and B). A closer examination of the adhered spores appeared to assemble in form of clusters, most likely caused by the drying process on the substrate (Figure 23C).

Figure 23. Adhesion of spores in solution at high spore concentration Images from a 5 ×10^7 spores/ml solution deposited on SMBP-HFBII coated glass slides (A) and an uncoated glass slides (B). After deposition and drying the slides were washed by immersion. The remaining, adhered, spores are observed in these images. At high concentrations above ~107 spores/ml, the spores tend to form aggregates (C). These aggregates adhere to SMBP-HFBII coated and uncoated glass slides in an uneven manner rendering dispersed adhesion results.

The adhesion of spores from highly concentrated solutions to coated and uncoated glass slides (Figure 24) showed a similar trend for both types of surfaces. The data was fit to a second order polynomial that plateaus approximately at 8 ×103 spores/mm2. This value was 30% lower than the estimated spore monolayer coverage of the surface (12 × 103 spores/mm2), suggesting that the loading capacity of both surfaces is limited by the spore concentration. Moreover, the spore adhesion to coated slides was consistently lower than the control indicating that the protein facilitated the washing of adhered spores when they were at high concentrations.

Figure 24. Effect of spore concentration on the spore adsorption to coated and uncoated glass The adsorption of spores in solution at pH 7.5 and different concentrations was tested on glass (circles) and SMBP-HFBII coated glass slides (triangles). The theoretical monolayer coverage (dashed line) was estimated assuming a monolayer of circular spores of 10 μm diameter. Left: images of glass substrates at different levels of surface saturation.

4.5 Discussion

On 2011, the Swedish Institute for Food and Biotechnology (SIK) and the Food and

Agriculture Organization of the United Nations (FAO) reported alarming levels of food losses of 45% for all the tubers, fruits and vegetables produced worldwide96. Despite current strategies to reduce their impact, microbes and particularly fungi still play important roles in food loss at all levels of the food supply chain106,110,111. B. cinerea is a

widespread fungal pathogen with enormous impact on fruits and flowers losses. In this work, we examined the interaction of B. cinerea spores with solid substrates as it is a required step for fungi-host interaction prior to infection and disease development.

The hydrophobicity of materials was confirmed as a primordial factor for the adhesion of air-dispersed, non-germinated spores to the materials surfaces (Figure 21). These results matched what have been reported previously for water-dispersed spores from B. cinerea and other fungi109,112. We also found that when spores are in solution, the pH has a notorious effect on their adhesion behaviors. This suggests that electrostatic interactions might have important roles in spore-substrate interactions too. Electrostatic interactions could be due to the modification of the spore wall components (proteins and sugars mainly). The charged spore walls would make the spores more hydrophilic and easier to wash off the surface.

The role of hydrophobins in spore hydrophobicity and adhesion has been found variable depending on the species studied22,113. In B. cinerea HFBs were not required for spore hydrophobicity but important for the fungal adhesion to substrates21. Therefore, we wanted to explore the effects of HFB coatings on the material-spore interaction and the possibility to modulate their adhesion. Our results with unsaturated surfaces show a slight increase of spore adhesion that depend on spore concentration and pH. At low spore concentrations the coating slightly increases their adhesion to glass. This effect is greater when the coating is crosslinked and at pH 5 (below SMBP-HFBII isoelectric point).

These observations suggest a possible rearrangement and leaking of the protein coating

that is inhibited by the crosslinking and the electrostatic interactions between the glass substrate (negatively charged) and the positive charges of the protein at pH 5.

Further research is required to optimize the effect of SMBP-HFBII on spore adhesion to hydrophilic substrates. Although our tests were carried out with fungal spores due to their important role in food decay our results are not constrained to specific hydrophobin- hydrophobin affinity interactions. Thus, this approach could be extended to other types of hydrophobic particles.

Chapter 5: Conclusions and further directions

Great interest has been given to biosurfactants due to their key roles in biological processes, technical applications, biocompatibility and high performance. Unfortunately, the high cost of production, low production yields and purification challenges have been a barrier for their use in industrial applications1,3,14,114. Hydrophobins have a remarkably high surface activity, unique self-assembling characteristics and stable structures that drive their behaviors. Their apparent simplicity and robust mechanisms invite to modify them at the genetic level for enhanced protein production and applications. Therefore, this work explored the design of engineered HFBs through a fusion strategy, their production, characterization and use as surface modifiers.

Producing HFBs and HFB fusion proteins presents three main drawbacks: (1) the low yields achieved by native producers and the lack of genetic tools to modify them, (2) the high toxicity of HFBs for heterologous hosts, which hinders the use of standard biotechnological expression pipelines, and (3) the purification challenges generated by their foaming and self-assembling. The first step of our work was the design of HFB fusion proteins to overcome these limitations.

We used HFBII as a model protein for the modulation of HFB properties via a fusion strategy. We tested the effect of several fusion partners in facilitating its expression and purification in E. coli. This host was chosen due to its genetic flexibility, fast growth rate and availability of tools for genetic manipulation. Gene silencing and inhibition of

bacterial growth showed that our fusion HFBs are highly toxic for the E. coli and that their toxicity correlates with the solubility of the proteins expressed. These effects could be palliated by different fusion partners and by modulating the growth and expression conditions. Our results showed that the fusion with SMBP, a small, robust and highly hydrophilic metalloprotein, allowed expression, increased product solubility and facilitated its rapid purification through non-denaturing liquid chromatography. This purification method is standard and compatible with a wider range of possible fusion targets. Therefore, our results are encouraging towards the use of small metalloproteins, like SMBP, for the construction of more diverse and complex protein systems to be used in a wider array of HFB applications.

Nonetheless, the gene expression was consistently silenced for all the HFB fusion proteins designed. This effect demonstrated that our bacterial host is not an optimal expression system. Gene silencing have also been reported in plants59. Furthermore, the production of inclusion bodies and low yields reported in bacteria45 suggest analogous results found by other research groups. Hence, it becomes evident the importance of building genetic tools that allow the overproduction of complex or toxic proteins such as

HFBs in their host organisms, where managing strategies are already in place. Alternative approaches to overcome this limitation are protein engineering solutions to decrease the inherent toxicity of HFBs for heterologous hosts.

Native HFBs have a robust and rapid self-assembling that limits their applicability, mostly when immobilized with standard coating techniques. Therefore, we explored the modulation of HFBII’s interfacial and self-assembling properties through a fusion

strategy. We have demonstrated that SMBP-HFBII shows a marked hydropathy change that increases the protein’s affinity for hydrophilic surfaces while preserving its surfactant behaviors. This result establishes the potential use of a fusion strategy for the enhancement of the native HFBs’.

Each HFB has differential properties such as hydrophobicity and self-assembling behaviors, which ultimately will determine its optimal applicability29. Our knowledge limitations hinder the optimal selection of the best proteins for precise applications. Thus, the rational HFB enhancement through fusion strategies is a powerful avenue to bridge our knowledge gaps on their sequence/structure/function relationships by fine-tune the native features of the already known proteins.

Further research in HFB production systems will be needed for general uses of this protein family. In our group, ongoing projects will continue exploring potential applications for fusion HBFs. In particular, we will examine the use of highly stable foams and nanobubbles generated by SMBP-HFBII for CO2 capturing technologies.

Additionally, the use of HFBs for surface functionalization will be explored to produce modular surfactant proteins with a better dispersion in multiphase systems such as plastic polymers and paintings92,115,116.

References

(1) Sunde, M.; Pham, C. L. L.; Kwan, A. H. Molecular Characteristics and Biological

Functions of Surface-Active and Surfactant Proteins. Annu. Rev. Biochem. 2017,

86 (1), 585–608.

(2) McClements, D. J.; Gumus, C. E. Natural Emulsifiers — Biosurfactants,

Phospholipids, Biopolymers, and Colloidal Particles: Molecular and

Physicochemical Basis of Functional Performance. Adv. Colloid Interface Sci.

2016, 234, 3–26.

(3) Banat, I. M.; Franzetti, A.; Gandolfi, I.; Bestetti, G.; Martinotti, M. G.; Fracchia,

L.; Smyth, T. J.; Marchant, R. Microbial Biosurfactants Production, Applications

and Future Potential. Appl. Microbiol. Biotechnol. 2010, 87 (2), 427–444.

(4) Schor, M.; Reid, J. L.; Macphee, C. E.; Stanley-wall, N. R. The Diverse Structures

and Functions of Surfactant Proteins. Trends Biochem. Sci. 2016, 41 (7), 610–620.

(5) Raaijmakers, J. M.; De Bruijn, I.; Nybroe, O.; Ongena, M. Natural Functions of

Lipopeptides from Bacillus and Pseudomonas : More than Surfactants and

Antibiotics. FEMS Microbiol. Rev. 2010, 34 (6), 1037–1062.

(6) Walsh, C. T.; Fischbach, M. A. Natural Products Version 2.0: Connecting Genes

to Molecules. J. Am. Chem. Soc. 2010, 132 (8), 2469–2493.

(7) Onaizi, S. A.; Nasser, M. S.; Twaiq, F. Adsorption and Thermodynamics of

Biosurfactant, Surfactin, Monolayers at the Air-Buffered Liquid Interface. Colloid

Polym. Sci. 2014, 292 (7), 1649–1656. 74

(8) Marin, C. P.; Kaschuk, J. J.; Frollini, E.; Nitschke, M. Potential Use of the Liquor

from Sisal Pulp Hydrolysis as Substrate for Surfactin Production. Ind. Crops Prod.

2015, 66, 239–245.

(9) Morris, R. J.; Bromley, K. M.; Stanley-Wall, N.; MacPhee, C. E. A

Phenomenological Description of BslA Assemblies across Multiple Length Scales.

Philos. Trans. R. Soc. A Math. Phys. Eng. Sci. 2016, 374 (2072), 20150131.

(10) Bromley, K. M.; Morris, R. J.; Hobley, L.; Brandani, G.; Gillespie, R. M. C.;

McCluskey, M.; Zachariae, U.; Marenduzzo, D.; Stanley-Wall, N. R.; MacPhee, C.

E. Interfacial Self-Assembly of a Bacterial Hydrophobin. Proc. Natl. Acad. Sci. U.

S. A. 2015, 112 (17), 5419–5424.

(11) Schenk, H. J.; Espino, S.; Romo, D. M.; Nima, N.; Do, A. Y. T.; Michaud, J. M.;

Papahadjopoulos-Sternberg, B.; Yang, J.; Zuo, Y. Y.; Steppe, K.; et al. Xylem

Surfactants Introduce a New Element to the Cohesion-Tension Theory. Plant

Physiol. 2017, 173 (2), 1177–1196.

(12) Pereira, L.; Flores-Borges, D.; Bittencourt, P.; Mayer, J.; Kiyota, E.; Araújo, P.;

Jansen, S.; Freitas, R.; Oliveira, R.; Mazzafera, P. Infrared Nanospectroscopy

Reveals the Chemical Nature of Pit Membranes in Water-Conducting Cells of the

Plant Xylem. Plant Physiol. 2018, pp.00138.2018.

(13) Linder, M. B. Hydrophobins: Proteins That Self Assemble at Interfaces. Curr.

Opin. Colloid Interface Sci. 2009, 14 (5), 356–363.

(14) Khalesi, M.; Gebruers, K.; Derdelinckx, G. Recent Advances in Fungal 75

Hydrophobin Towards Using in Industry. Protein J. 2015, 34 (4), 243–255.

(15) Mulder, G. H.; Wessels, J. G. H. Molecular Cloning of RNAs Differentially

Expressed in Monokaryons and Dikaryons OfSchizophyllum Commune in

Relation to Fruiting. Exp. Mycol. 1986, 10 (3), 214–227.

(16) Wessels, J. G. H.; De Vries, O. M. H.; Ásgeirsdóttir, S. A.; Schuren, F. H. J.

Hydrophobin Genes Lnvolved in Formation of Aerial Hyphae and Fruit Bodies in

Schizophyllum. Plant Cell 1991, 3, 793–799.

(17) Nakari-Setälä, T.; Aro, N.; Ilmén, M.; Muñoz, G.; Kalkkinen, N.; Penttilä, M.

Differential Expression of the Vegetative and Spore-Bound Hydrophobins of

Trichoderma Reesei: Cloning and Characterization of the Hfb2 Gene. VTT Publ.

1996, 423, 1–15.

(18) Terhem, R. B.; van Kan, J. A. L. Functional Analysis of Hydrophobin Genes in

Sexual Development of Botrytis Cinerea. Fungal Genet. Biol. 2014, 71, 42–51.

(19) Khalesi, M.; Zune, Q.; Telek, S.; Riveros-Galan, D.; Verachtert, H.; Toye, D.;

Gebruers, K.; Derdelinckx, G.; Delvigne, F. Fungal Biofilm Reactor Improves the

Productivity of Hydrophobin HFBII. Biochem. Eng. J. 2014, 88, 171–178.

(20) Wösten, H. A. B.; Scholtmeijer, K. Applications of Hydrophobins: Current State

and Perspectives. Appl. Microbiol. Biotechnol. 2015, 99 (4), 1587–1597.

(21) Izumitsu, K.; Kimura, S.; Kobayashi, H.; Morita, A.; Saitoh, Y.; Tanaka, C. Class I

Hydrophobin BcHpb1 Is Important for Adhesion but Not for Later Infection of

Botrytis Cinerea. J. Gen. Plant Pathol. 2010, 76 (4), 254–260.

(22) Dubey, M. K.; Jensen, D. F.; Karlsson, M. Hydrophobins Are Required for

Conidial Hydrophobicity and Plant Root Colonization in the Fungal Biocontrol

Agent Clonostachys Rosea. BMC Microbiol. 2014, 14, 18.

(23) Takahashi, T.; Maeda, H.; Yoneda, S.; Ohtaki, S.; Yamagata, Y.; Hasegawa, F.;

Gomi, K.; Nakajima, T.; Abe, K. The Fungal Hydrophobin RolA Recruits

Polyesterase and Laterally Moves on Hydrophobic Surfaces. Mol. Microbiol.

2005, 57 (6), 1780–1796.

(24) Lacroix, H.; Whiteford, J. R.; Spanu, P. D. Localization of Cladosporium Fulvum

Hydrophobins Reveals a Role for HCf-6 in Adhesion. FEMS Microbiol. Lett.

2008, 286 (1), 136–144.

(25) Huang, Y.; Mijiti, G.; Wang, Z.; Yu, W.; Fan, H.; Zhang, R.; Liu, Z. Functional

Analysis of the Class II Hydrophobin Gene HFB2-6 from the Biocontrol Agent

Trichoderma Asperellum ACCC30536. Microbiol. Res. 2015, 171, 8–20.

(26) Kyte, J.; Doolittle, R. F. A Simple Method for Displaying the Hydropathic

Character of a Protein. J. Mol. Biol. 1982, 157 (1), 105–132.

(27) Askolin, S.; Linder, M.; Scholtmeijer, K.; Tenkanen, M.; Penttilä, M.; De Vocht,

M. L.; Wö Sten, H. A. B. Interaction and Comparison of a Class I Hydrophobin

from Schizophyllum Commune and Class II Hydrophobins from Trichoderma

Reesei. Bioma 2006, 7, 1295–1301.

(28) Stanimirova, R. D.; Gurkov, T. D.; Kralchevsky, P. A.; Balashev, K. T.; Stoyanov,

S. D.; Pelan, E. G. Surface Pressure and Elasticity of Hydrophobin HFBII Layers

on the Air–Water Interface: Rheology Versus Structure Detected by AFM

Imaging. Langmuir 2013, 29 (20), 6053–6067.

(29) Zampieri, F.; Wösten, H. A. B.; Scholtmeijer, K. Creating Surface Properties

Using a Palette of Hydrophobins. Materials (Basel). 2010, 3 (9), 4607–4625.

(30) Sallada, N. D.; Dunn, K. J.; Berger, B. W. A Structural and Functional Role for

Disulfide Bonds in a Class II Hydrophobin. Biochemistry 2018, 57 (5), 645–653.

(31) Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M. R.; Appel, R.

D.; Bairoch, A. Protein Identification and Analysis Tools on the ExPASy Server.

In The Proteomics Protocols Handbook; Humana Press: Totowa, NJ, 2005; pp

571–607.

(32) Hakanpää, J.; Paananen, A.; Askolin, S.; Nakari-Setälä, T.; Parkkinen, T.; Penttilä,

M.; Linder, M. B.; Rouvinen, J. Atomic Resolution Structure of the HFBII

Hydrophobin, a Self-Assembling Amphiphile. J. Biol. Chem. 2004, 279 (1), 534–

539.

(33) Hakanpää, J.; Linder, M.; Popov, A.; Schmidt, A.; Rouvinen, J. Hydrophobin

HFBII in Detail: Ultrahigh-Resolution Structure at 0.75 Å. Acta Crystallogr. Sect.

D Biol. Crystallogr. 2006, 62 (4), 356–367.

(34) Askolin, S.; Linder, M.; Scholtmeijer, K.; Tenkanen, M.; Penttilä, M.; de Vocht,

M. L.; Wösten, H. A. B. Interaction and Comparison of a Class I Hydrophobin 78

from Schizophyllum Commune and Class II Hydrophobins Trichoderma Reesei.

Biomacromolecules 2006, 7 (4), 1295–1301.

(35) Kallio, J. M.; Linder, M. B.; Rouvinen, J. Crystal Structures of Hydrophobin

HFBII in the Presence of Detergent Implicate the Formation of Fibrils and

Monolayer Films. J. Biol. Chem. 2007, 282 (39), 28733–28739.

(36) Ritva, S.; Torkkeli, M.; Paananen, A.; Linder, M.; Kisko, K.; Knaapila, M.; Ikkala,

O.; Vuorimaa, E.; Lemmetyinen, H.; Seeck, O. Self-Assembled Structures of

Hydrophobins HFBI and HFBII. J. Appl. Crystallogr. 2003, 36 (3), 499–502.

(37) Cox, A. R.; Aldred, D. L.; Russell, A. B. Exceptional Stability of Food Foams

Using Class II Hydrophobin HFBII. Food Hydrocoll. 2009, 23 (2), 366–376.

(38) Winterburn, J. B.; Russell, A. B.; Martin, P. J. Characterisation of HFBII

Biosurfactant Production and Foam Fractionation with and without Antifoaming

Agents. Appl. Microbiol. Biotechnol. 2011, 90 (3), 911–920.

(39) Wang, K.; Xiao, Y.; Wang, Y.; Feng, Y.; Chen, C.; Zhang, J.; Zhang, Q.; Meng,

S.; Wang, Z.; Yang, H.; et al. Self-Assembled Hydrophobin for Producing Water-

Soluble and Membrane Permeable Fluorescent Dye. Sci. Rep. 2016, 6, 23061.

(40) Kurppa, K.; Jiang, H.; Szilvay, G. R.; Nasibulin, A. G.; Kauppinen, E. I.; Linder,

M. B. Controlled Hybrid Nanostructures through Protein-Mediated Noncovalent

Functionalization of Carbon Nanotubes. Angew. Chemie Int. Ed. 2007, 46 (34),

6446–6449.

(41) Fokina, O.; Fenchel, A.; Winandy, L.; Fischer, R. Immobilization of LccC Laccase

from Aspergillus Nidulans on Hard Surfaces via Fungal Hydrophobins. Appl.

Environ. Microbiol. 2016, 82 (21), 6395–6402.

(42) Espino-Rammer, L.; Ribitsch, D.; Przylucka, A.; Marold, A.; Greimel, K. J.;

Herrero Acero, E.; Guebitz, G. M.; Kubicek, C. P.; Druzhinina, I. S. Two Novel

Class II Hydrophobins from Trichoderma Spp. Stimulate Enzymatic Hydrolysis of

Poly(Ethylene Terephthalate) When Expressed as Fusion Proteins. Appl. Environ.

Microbiol. 2013, 79 (14), 4230–4238.

(43) Qin, M.; Hou, S.; Wang, L.; Feng, X.; Wang, R.; Yang, Y.; Wang, C.; Yu, L.;

Shao, B.; Qiao, M. Two Methods for Glass Surface Modification and Their

Application in Protein Immobilization. Colloids Surfaces B Biointerfaces 2007, 60,

243–249.

(44) De Stefano, L.; Rea, I.; De Tommasi, E.; Rendina, I.; Rotiroti, L.; Giocondo, M.;

Longobardi, S.; Armenante, A.; Giardina, P. Bioactive Modification of Silicon

Surface Using Self-Assembled Hydrophobins from Pleurotus Ostreatus. Eur. Phys.

J. E 2009, 30 (2), 181–185.

(45) Kirkland, B. H.; Keyhani, N. O. Expression and Purification of a Functionally

Active Class i Fungal Hydrophobin from the Entomopathogenic Fungus Beauveria

Bassiana in E. Coli. J. Ind. Microbiol. Biotechnol. 2011, 38 (2), 327–335.

(46) Reuter, L. J.; Bailey, M. J.; Joensuu, J. J.; Ritala, A. Scale-up of Hydrophobin-

Assisted Recombinant Protein Production in Tobacco BY-2 Suspension Cells.

Plant Biotechnol. J. 2014, 12 (4), 402–410.

(47) Reuter, L.; Ritala, A.; Linder, M.; Joensuu, J.; Hornyik, C.; Tavazza, M. Novel

Hydrophobin Fusion Tags for Plant-Produced Fusion Proteins. PLoS One 2016, 11

(10), e0164032.

(48) Penttilä, M.; NAKARI-SETÄLÄ, T.; FAGERSTRÖM, R.; Selber, K.; Kula, M.-

R.; Linder, M.; Tjerneld, F. Process for Partitioning of Proteins. WO2000058342

A1, 2000.

(49) Kurppa, K.; Reuter, L. J.; Ritala, A.; Linder, M. B.; Joensuu, J. J. In-Solution

Antibody Harvesting with a Plant-Produced Hydrophobin-Protein A Fusion. Plant

Biotechnol. J. 2018, 16 (2), 404–414.

(50) Wang, X.; Mao, J.; Chen, Y.; Song, D.; Gao, Z.; Zhang, X.; Bai, Y.; Saris, P. E. J.;

Feng, H.; Xu, H.; et al. Design of Antibacterial Biointerfaces by Surface

Modification of Poly (ε-Caprolactone) with Fusion Protein Containing

Hydrophobin and PA-1. Colloids Surfaces B Biointerfaces 2017, 151.

(51) Misra, R.; Li, J.; Cannon, G. C.; Morgan, S. E. Nanoscale Reduction in Surface

Friction of Polymer Surfaces Modified with Sc3 Hydrophobin from Schizophyllum

Commune. Biomacromolecules 2006, 7 (5), 1463–1470.

(52) Janssen, M. I.; van Leeuwen, M. B. M.; Scholtmeijer, K.; van Kooten, T. G.;

Dijkhuizen, L.; Wosten H.A.B. Coating with Genetic Engineered Hydrophobin

Promotes Growth of Fibroblasts on a Hydrophobic Solid. Biomaterials 2002, 23,

4847–4854. 81

(53) Gruner, L. J.; Ostermann, K.; Rödel, G. Layer Thickness of Hydrophobin Films

Leads to Oscillation in Wettability. Langmuir 2012, 28 (17), 6942–6949.

(54) Magarkar, A.; Mele, N.; Abdel-Rahman, N.; Butcher, S.; Torkkeli, M.; Serimaa,

R.; Paananen, A.; Linder, M.; Bunker, A. Hydrophobin Film Structure for HFBI

and HFBII and Mechanism for Accelerated Film Formation. PLoS Comput. Biol.

2014, 10 (7), e1003745.

(55) Kisko, K.; Torkkeli, M.; Vuorimaa, E.; Lemmetyinen, H.; Seeck, O. H.; Linder,

M.; Serimaa, R. Langmuir-Blodgett Films of Hydrophobins HFBI and HFBII. In

Surface Science; 2005; Vol. 584, pp 35–40.

(56) Szilvay, G. R.; Paananen, A.; Laurikainen, K.; Vuorimaa, E.; Lemmetyinen, H.;

Peltonen, J.; Linder, M. B. Self-Assembled Hydrophobin Protein Films at the

Air−Water Interface: Structural Analysis and Molecular Engineering †.

Biochemistry 2007, 46 (9), 2345–2354.

(57) Wohlleben, W.; Subkowski, T.; Bollschweiler, C.; von Vacano, B.; Liu, Y.;

Schrepp, W.; Baus, U. Recombinantly Produced Hydrophobins from Fungal

Analogues as Highly Surface-Active Performance Proteins. Eur. Biophys. J. 2010,

39 (3), 457–468.

(58) Gutiérrez, S. P.; Saberianfar, R.; Kohalmi, S. E.; Menassa, R. Protein Body

Formation in Stable Transgenic Tobacco Expressing Elastin-like Polypeptide and

Hydrophobin Fusion Proteins. BMC Biotechnol. 2013, 13, 40.

(59) Joensuu, J. J.; Conley, A. J.; Lienemann, M.; Brandle, J. E.; Linder, M. B.; 82

Menassa, R. Hydrophobin Fusions for High-Level Transient Protein Expression

and Purification in Nicotiana Benthamiana. PLANT Physiol. 2010, 152 (2), 622–

633.

(60) Lienemann, M.; Grune, M. S.; Paananen, A.; Siika-aho, M.; Linder, M. B. Charge-

Based Engineering of Hydrophobin HFBI: E Ff Ect on Interfacial Assembly and

Interactions. 2015.

(61) M., B.; S., A.; N., H.; M., T.; M., L.; M., P.; T., N.-S. Process Technological

Effects of Deletion and Amplification of Hydrophobins I and II in Transformants

of Trichoderma Reesei. Appl. Microbiol. Biotechnol. 2002, 58 (6), 721–727.

(62) Song, D.; Gao, Z.; Zhao, L.; Wang, X.; Xu, H.; Bai, Y.; Zhang, X.; Linder, M. B.;

Feng, H.; Qiao, M. High-Yield Fermentation and a Novel Heat-Precipitation

Purification Method for Hydrophobin HGFI from Grifola Frondosa in Pichia

Pastoris. Protein Expr. Purif. 2016, 128.

(63) Barney, B. M.; LoBrutto, R.; Francisco, W. A. Characterization of a Small Metal

Binding Protein from Nitrosomonas Europaea. Biochemistry 2004, 43 (35),

11206–11213.

(64) Khalesi, M.; Venken, T.; Deckers, S.; Winterburn, J.; Shokribousjein, Z.;

Gebruers, K.; Verachtert, H.; Delcour, J.; Martin, P.; Derdelinckx, G. A Novel

Method for Hydrophobin Extraction Using CO 2 Foam Fractionation System. Ind.

Crop. Prod. 2013, 43, 372–377.

(65) Kupčík, R.; Zelená, M.; Řehulka, P.; Bílková, Z.; Česlová, L. Selective Isolation of 83

Hydrophobin SC3 by Solid-Phase Extraction with Polytetrafluoroethylene

Microparticles and Subsequent Mass Spectrometric Analysis. J. Sep. Sci. 2016, 39

(4), 717–724.

(66) Asakawa, H.; Tahara, S.; Nakamichi, M.; Takehara, K.; Ikeno, S.; Linder, M. B.;

Haruyama, T. The Amphiphilic Protein HFBII as a Genetically Taggable

Molecular Carrier for the Formation of a Self-Organized Functional Protein Layer

on a Solid Surface. Langmuir 2009, 25 (16), 8841–8844.

(67) Saha, R. P.; Samanta, S.; Patra, S.; Sarkar, D.; Saha, A.; Singh, M. K. Metal

Homeostasis in Bacteria: The Role of ArsR–SmtB Family of Transcriptional

Repressors in Combating Varying Metal Concentrations in the Environment.

BioMetals 2017, 30 (4), 459–503.

(68) Hirota, S.; Lin, Y.-W. Design of Artificial Metalloproteins/Metalloenzymes by

Tuning Noncovalent Interactions. JBIC J. Biol. Inorg. Chem. 2018, 23 (1), 7–25.

(69) Stothard, P. The Sequence Manipulation Suite: JavaScript Programs for Analyzing

and Formatting Protein and DNA Sequences. Biotechniques 2000, 28 (6), 1102–

1104.

(70) Chen, X.; Wang, Y.; Wang, P. Peptide-Induced Affinity Binding of Carbonic

Anhydrase to Carbon Nanotubes. Langmuir 2015, 31 (1), 397–403.

(71) Dai, G.; Hu, J.; Zhao, X.; Wang, P. A Colorimetric Paper Sensor for Lactate Assay

Using a Cellulose-Binding Recombinant Enzyme. Sensors Actuators B Chem.

2017, 238 (238), 138–144. 84

(72) Wilkins, M. R.; Gasteiger, E.; Bairoch, A.; Sanchez, J. C.; Williams, K. L.; Appel,

R. D.; Hochstrasser, D. F. Protein Identification and Analysis Tools in the ExPASy

Server. Methods Mol. Biol. 1999, 112, 531–552.

(73) Zhang, X. L.; Penfold, J.; Thomas, R. K.; Tucker, I. M.; Petkov, J. T.; Bent, J.;

Cox, A.; Grillo, I. Self-Assembly of Hydrophobin and Hydrophobin/Surfactant

Mixtures in Aqueous Solution. Langmuir 2011, 27 (17), 10514–10522.

(74) Zhi, Y.; Wu, Q.; Xu, Y. Production of Surfactin from Waste Distillers’ Grains by

Co-Culture Fermentation of Two Bacillus Amyloliquefaciens Strains. Bioresour.

Technol. 2017, 235, 96–103.

(75) Radzuan, M. N.; Banat, I. M.; Winterburn, J. Production and Characterization of

Rhamnolipid Using Palm Oil Agricultural Refinery Waste. Bioresour. Technol.

2017, 225, 99–105.

(76) Kolb, A.; Busby, S.; Bue, H.; Garges, S.; Adhya, S. TRANSCRIPTIONAL

REGULATION BY CAMP AND ITS RECEPTOR PROTEIN L; 1993; Vol. 62.

(77) Derdelinckx. Combined Modeling and Biophysical Characterisation of CO(2)

Interaction with Class II Hydrophobins: New Insight into the Mechanism

Underpinning Primary Gushing. J. Am. Soc. Brew. Chem. 2012.

(78) Lutterschmid, G.; Stübner, M.; Vogel, R. F.; Niessen, L. Induction of Gushing

with Recombinant Class II Hydrophobin FcHyd5p from Fusarium Culmorum and

the Impact of Hop Compounds on Its Gushing Potential. J. Inst. Brew. 2010, 116

(4), 339–347. 85

(79) Torkkeli, M.; Serimaa, R.; Ikkala, O.; Linder, M. Aggregation and Self-Assembly

of Hydrophobins from Trichoderma Reesei: Low-Resolution Structural Models.

Biophys. J. 2002, 83 (4), 2240–2247.

(80) Kisko, K.; Szilvay, G. R.; Vainio, U.; Linder, M. B.; Serimaa, R. Interactions of

Hydrophobin Proteins in Solution Studied by Small-Angle X-Ray Scattering.

Biophys. J. 2008, 94 (1), 198–206.

(81) Lienemann, M.; Gandier, J.-A.; Joensuu, J. J.; Iwanaga, A.; Takatsuji, Y.;

Haruyama, T.; Master, E.; Tenkanen, M.; Linder, M. B. Structure-Function

Relationships in Hydrophobins: Probing the Role of Charged Side Chains. Appl.

Environ. Microbiol. 2013, 79 (18), 5533–5538.

(82) Basheva, E. S.; Kralchevsky, P. A.; Danov, K. D.; Stoyanov, S. D.; Blijdenstein,

T. B. J.; Pelan, E. G.; Lips, A. Self-Assembled Bilayers from the Protein HFBII

Hydrophobin: Nature of the Adhesion Energy. Langmuir 2011, 27 (8), 4481–4488.

(83) Cox, A. R.; Cagnol, F.; Russell, A. B.; Izzard, M. J. Surface Properties of Class II

Hydrophobins from Trichoderma Reesei and Influence on Bubble Stability.

Langmuir 2007, 23 (15), 7995–8002.

(84) Alexandrov, N. A.; Marinova, K. G.; Gurkov, T. D.; Danov, K. D.; Kralchevsky,

P. A.; Stoyanov, S. D.; Blijdenstein, T. B. J.; Arnaudov, L. N.; Pelan, E. G.; Lips,

A. Interfacial Layers from the Protein HFBII Hydrophobin: Dynamic Surface

Tension, Dilatational Elasticity and Relaxation Times. J. Colloid Interface Sci.

2012, 376 (1), 296–306.

(85) Askolin, S.; Linder, M.; Scholtmeijer, K.; Tenkanen, M.; Penttila, M.; Vocht, M.

L. De; Wo, H. A. B. Interaction and Comparison of a Class I Hydrophobin from

Schizophyllum Commune and Class II Hydrophobins from Trichoderma Reesei.

2006, 1295–1301.

(86) Kim, J.; Grate, J. W.; Wang, P. Nanostructures for Enzyme Stabilization. Chem.

Eng. Sci. 2006, 61 (3), 1017–1026.

(87) Sheldon, R. A.; van Pelt, S. Enzyme Immobilisation in Biocatalysis: Why, What

and How. Chem. Soc. Rev. 2013, 42 (15), 6223–6235.

(88) Linder, M.; Szilvay, G. R.; Nakari-Setälä, T.; Söderlund, H.; Penttilä, M. Surface

Adhesion of Fusion Proteins Containing the Hydrophobins HFBI and HFBII from

Trichoderma Reesei. Protein Sci. 2002, 11 (9), 2257–2266.

(89) Magarkar, A.; Mele, N.; Abdel-Rahman, N.; Butcher, S.; Torkkeli, M.; Serimaa,

R.; Paananen, A.; Linder, M.; Bunker, A. Hydrophobin Film Structure for HFBI

and HFBII and Mechanism for Accelerated Film Formation. PLoS Comput. Biol.

2014, 10 (7), e1003745.

(90) Wagner, M. S.; Mcarthur, S. L.; Shen, M.; Horbett, T. A.; Castner, D. G.; Wagner,

M. S.; Mcarthur, S. L.; Shen, M.; Thomas, A. Limits of Detection for Time of

Flight Secondary Ion Mass Spectrometry ( ToF-SIMS ) and X-Ray Photoelectron

Spectroscopy ( XPS ): Detection of Low Amounts of Adsorbed Protein. J.

Biomater. Sci. Polym. Edn. 2002, 13 (4), 407–428.

(91) Behrens, S. H.; Grier, D. G. The Charge of Glass and Silica Surfaces. J. Chem. 87

Phys 2001, 115, 6716–6721.

(92) Zhang, L.; Wu, S.; Buthe, A.; Zhao, X.; Jia, H.; Zhang, S.; Wang, P. Poly(Ethylene

Glycol) Conjugated Enzyme with Enhanced Hydrophobic Compatibility for Self-

Cleaning Coatings. ACS Appl. Mater. Interfaces 2012, 4 (11), 5981–5987.

(93) Grunér, M. S.; Szilvay, G. R.; Berglin, M.; Lienemann, M.; Laaksonen, P.; Linder,

M. B. Self-Assembly of Class II Hydrophobins on Polar Surfaces. Langmuir 2012,

28 (9), 4293–4300.

(94) Dean, R.; Van Kan, J. A. L.; Pretorius, Z. A.; Hammond-Kosack, K. E.; Pietro, A.

Di; Spanu, P. D.; Rudd, J. J.; Dickman, M.; Kahmann, R.; Ellis, J.; et al. The Top

10 Fungal Pathogens in Molecular Plant Pathology. Mol. Plant Pathol. 2012, 414–

430.

(95) Williamson, B.; Duncan ’, G. H.; Harrison1, J. G.; Harding, L. A.; Elad, Y.;

Zimand3, G. Effect of Humidity on Infection of Rose Petals by Dry-Inoculated

Conidia of Botrytis Cinerea. Mycol. Res. 1995, 99 (11), 1303–1310.

(96) Gustavsson, J.; Cederberg, C.; Sonesson, U. Global Food Losses and Food Waste -

Extent, Causes and Prevention; 2011.

(97) Kranz, J.; Rotem, J. Experimental Techniques in Plant Disease Epidemiology, 1st

ed.; Kranz, J., Rotem, J., Eds.; 1988.

(98) Williamson, B.; Tudzynski, B.; Tudzynski, P.; Van Kan, J. A. L. Botrytis Cinerea :

The Cause of Grey Mould Disease. Mol. Plant Pathol. 2007, 8 (5), 561–580.

(99) Jarvis, W. R. The Dispersal of Spores of Botrytis Cinerea Fr. in a Raspberry

Plantation. Trans. Br. Mycol. Soc. 1962, 45 (4), 549–559.

(100) Botrytis: Biology, Pathology and Control; Elad, Y., Williamson, B., Tudzynski, P.,

Delen, N., Eds.; Springer, 2007.

(101) Amselem, J.; Cuomo, C. A.; L van Kan, J. A.; Viaud, M.; Benito, E. P.; Couloux,

A.; Coutinho, P. M.; de Vries, R. P.; Dyer, P. S.; Fillinger, S.; et al. Genomic

Analysis of the Necrotrophic Fungal Pathogens Sclerotinia Sclerotiorum and

Botrytis Cinerea. PLoS Genet. | www.plosgenetics.org 1 2011, 7 (8).

(102) Fernández-Ortuño, D.; Grabke, A.; Li, X.; Schnabel, G. Independent Emergence of

Resistance to Seven Chemical Classes of Fungicides in Botrytis Cinerea.

Phytopathology 2015, 105 (4), 424–432.

(103) Liu, S.; Che, Z.; Chen, G. Multiple-Fungicide Resistance to Carbendazim,

Diethofencarb, Procymidone, and Pyrimethanil in Field Isolates of Botrytis

Cinerea from Tomato in Henan Province, China. Crop Prot. 2016, 84, 56–61.

(104) Hu, M.-J.; Fernández-Ortuño, D.; Schnabel, G. Monitoring Resistance to SDHI

Fungicides in Botrytis Cinerea From Strawberry Fields. Plant Dis. 2016, 100 (5),

959–965.

(105) Leroch, M.; Plesken, C.; Weber, R. W. S.; Kauff, F.; Scalliet, G.; Hahn, M. Gray

Mold Populations in German Strawberry Fields Are Resistant to Multiple

Fungicides and Dominated by a Novel Clade Closely Related to Botrytis Cinerea.

Appl. Environ. Microbiol. 2013, 79 (1), 159–167. 89

(106) Filonow, A. B. A Procedure for Quantifying Adhesion of Conidia of Botrytis

Cinerea to the Skin of Apple Fruit.

(107) Kennedy, R.; Wakeham, A. J.; Byrne, K. G.; Meyer, U. M.; Dewey, F. M. A New

Method To Monitor Airborne Inoculum of the Fungal Plant Pathogens

Mycosphaerella Brassicicola and Botrytis Cinerea. Appl. Environ. Microbiol.

2000, 66 (7), 2996–3000.

(108) Vu, K. D.; Hollingsworth, R. G.; Leroux, E.; Salmieri, S.; Lacroix, M.

Development of Edible Bioactive Coating Based on Modified Chitosan for

Increasing the Shelf Life of Strawberries. Food Res. Int. 2011, 44 (1), 198–203.

(109) Doss, R. P.; Potier, S. W.; Chastagner, G. A.; Christian ’, J. K. Adhesion of

Nongerminated Botrytis Cinerea Conidia to Several Substratat. Appl. Environ.

Microbiol. 1993, 1786–1791.

(110) Gerbeaud, C.; Giermanska-Kahn, J.; Meleard, P.; Pouligny, B.; Latorse, M.-P.

Using Capillary Forces to Estimate the Adhesion Strength of Magnaporthe Grisea

Spores on Glass. Comptes Rendus l’Académie des Sci. - Ser. IV - Phys. 2001, 2 (8),

1235–1240.

(111) Su’udi, M.; Kim, J.; Park, J.-M.; Bae, S.-C.; Kim, D.; Kim, Y.-H.; Ahn, I.-P.

Quantification of Rice Blast Disease Progressions through Taqman Real-Time

PCR. Mol. Biotechnol. 2013, 55 (1), 43–48.

(112) Newey, L. J.; Caten, C. E.; Green, J. R. Rapid Adhesion of Stagonospora Nodorum

Spores to a Hydrophobic Surface Requires Pre-Formed Cell Surface 90

Glycoproteins. Mycol. Res. 2007, 111 (Pt 11), 1255–1267.

(113) Mosbach, A.; Leroch, M.; Mendgen, K. W.; Hahn, M. Lack of Evidence for a Role

of Hydrophobins in Conferring Surface Hydrophobicity to Conidia and Hyphae of

Botrytis Cinerea. BMC Microbiol. 2011, 11, 10.

(114) Cheung, D.; Samantray, S.; Cheung, D. L.; Samantray, S. Molecular Dynamics

Simulation of Protein Biosurfactants. Colloids and Interfaces 2018, 2 (3), 39.

(115) Qin, M.; Wang, L.-K.; Feng, X.-Z.; Yang, Y.-L.; Wang, R.; Wang, C.; Yu, L.;

Shao, B.; Qiao, M.-Q. Bioactive Surface Modification of Mica and

Poly(Dimethylsiloxane) with Hydrophobins for Protein Immobilization. Langmuir

2007, 23 (8), 4465–4471.

(116) Jia, H.; Zhu, G.; Vugrinovich, B.; Kataphinan, W.; Reneker, D. H.; Wang, P.

Enzyme-Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as

Unique Biocatalysts. 2002, 1027–1032.