Pichia Pastoris Is a Suitable Host for the Heterologous Expression of Predicted Class I and Class II Hydrophobins for Discovery
Total Page：16
File Type：pdf, Size：1020Kb
microorganisms Article Pichia pastoris is a Suitable Host for the Heterologous Expression of Predicted Class I and Class II Hydrophobins for Discovery, Study, and Application in Biotechnology Julie-Anne Gandier * and Emma R. Master Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON MSE 3E5, Canada; [email protected] * Correspondence: [email protected]; Tel.: +1-416-946-7861 Received: 13 November 2017; Accepted: 29 December 2017; Published: 5 January 2018 Abstract: The heterologous expression of proteins is often a crucial first step in not only investigating their function, but also in their industrial application. The functional assembly and aggregation of hydrophobins offers intriguing biotechnological applications from surface modification to drug delivery, yet make developing systems for their heterologous expression challenging. In this article, we describe the development of Pichia pastoris KM71H strains capable of solubly producing the full set of predicted Cordyceps militaris hydrophobins CMil1 (Class IA), CMil2 (Class II), and CMil3 (IM) at mg/L yields with the use of 6His-tags not only for purification but for their detection. This result further demonstrates the feasibility of using P. pastoris as a host organism for the production of hydrophobins from all Ascomycota Class I subdivisions (a classification our previous work defined) as well as Class II. We highlight the specific challenges related to the production of hydrophobins, notably the challenge in detecting the protein that will be described, in particular during the screening of transformants. Together with the literature, our results continue to show that P. pastoris is a suitable host for the soluble heterologous expression of hydrophobins with a wide range of properties. Keywords: hydrophobin; Pichia pastoris; immunoassay; protein expression; fermentation; Cordyceps militaris 1. Introduction Filamentous fungi thrive in a wide range of environments throughout their life cycle. This ability can be attributed not only to the plasticity of their metabolism, but also to the often-overlooked non-catalytic proteins that are secreted and implicated in their adaptations to the surrounding environment. As the primary decomposers of all terrestrial ecosystems [1], these organisms produce enzymes that have been extensively studied for their ability to convert lignocellulosic biomass to soluble sugars for biofuels and biochemicals applications. The importance of non-catalytic, or weakly hydrolytic, accessory proteins in this process has also been considered. For instance, swollenins [2–4], cerato-platanins [5], and loosenins [6] have been implicated in cellulose disruption, which can increase accessibility to enzymes and improve conversion. There is increasing interest in screening non-catalytic fungal proteins for a broader range of applications. For instance, exclusively found in filamentous fungi of the Ascomycota and Basidiomycota phyla, hydrophobin proteins self-assemble to tailor interfaces to fungal life. These small 7–20 kDa proteins associate to coat and reverse the wettability of air-exposed fungal surfaces, such as spores [7,8] and fruiting bodies [9], serving roles in protection and adhesion [10]. They assemble at the air–liquid interface to reduce the surface tensions of liquid media allowing for the emergence of Microorganisms 2018, 6, 3; doi:10.3390/microorganisms6010003 www.mdpi.com/journal/microorganisms Microorganisms 2018, 6, 3 2 of 20 Microorganisms 2018, 6, 3 2 of 20 assemble at the air–liquid interface to reduce the surface tensions of liquid media allowing for the emergence of aerial structures [11]. In pathogenic fungi, they have been shown to act as virulence aerialfactors structures assembling [11]. on In pathogenicthe host’s fungi,surface they to haverecruit been enzymes, shown to facilitate act as virulence fungal factorsadhesion, assembling and/or ongenerate the host’s the surfacehydrophobic to recruit surface- enzymes,signal facilitate necessary fungal for the adhesion, development and/or of generate the infection the hydrophobic structures surface-signal[12]. While a wide necessary range for of theroles development has been propos of theed, infection the underlying structures feature [12]. Whileshared a amongst wide range these of rolesproteins has is been interfacial proposed, assembly. the underlying feature shared amongst these proteins is interfacial assembly. TheseThese biologicalbiological functionsfunctions have inspiredinspired aa numbernumber ofof biotechnologicalbiotechnological applications,applications, whichwhich areare currentlycurrently atat variousvarious stagesstages ofof developmentdevelopment (reviewed(reviewed inin [[13–15]).13–15]). Their uses have been explored for thethe dispersaldispersal ofof hydrophobichydrophobic solids,solids, liquids,liquids, andand airair inin applicationsapplications suchsuch asas foodfood foamfoam stabilizationstabilization andand drugdrug deliverydelivery [ 16[16–19].–19]. Certain Certain hydrophobins hydrophobins have have been been used used in in the th laboratorye laboratory to coatto coat surfaces surfaces for thefor selectivethe selective immobilization immobilization of cells of cells [20] and[20] molecules,and molecules, such such as protein as protein [21]. Conversely,[21]. Conversely, others others have beenhave appliedbeen applied as antifouling as antifouling films [22films]. These [22]. seeminglyThese seemingly contradictory contradictory abilities abilities highlight highlight the palette the ofpalette surface of propertiessurface properties that can that be producedcan be produc dependinged depending on the choice on the of choice hydrophobin of hydrophobin and solution and conditionssolution conditions for a given for application. a given application. MostMost broadly, thisthis palettepalette cancan bebe divideddivided intointo twotwo classesclasses (I(I andand II)II) withwith distinctdistinct biophysicalbiophysical propertiesproperties andand sequence sequence features. features. Class Class I hydophobins I hydophob self-assembleins self-assemble at interfaces at interfaces to form to highlyform highly stable filmsstable that films can that only can be only disassociated be disassociated by strong by strong acids, suchacids, as such trifluoroacetic as trifluoroacetic acid or acid formic or formic acid. These acid. filmsThese take films on take a regular on a regular “rodlet” “rodlet” morphology morphology with anwith amyloid-like an amyloid-like structure. structure. Class Class II assemblies II assemblies are comparativelyare comparatively less less stable stable and and can becan disassociated be disassociated by detergent-alcohol by detergent-alcohol mixtures mixtures [23]. [23]. ConsideredConsidered together, thethe primaryprimary sequencessequences belongingbelonging to thethe hydrophobinhydrophobin familyfamily shareshare littlelittle similaritysimilarity exceptexcept forfor aa conservedconserved motifmotif ofof eighteight cysteinescysteines withwith characteristiccharacteristic disulfidedisulfide bridgingbridging (Figure(Figure1 1))[ [24].24]. Similarity Similarity emerges emerges when when examining examining hydrophobin hydrophobin classes classes individually. individually. While While Class Class II IIsequences sequences are are relatively relatively well-conserved, well-conserved, it itis isin in fact fact Class Class I Ihydrophobins hydrophobins that that share share little to nono similaritysimilarity despite despite the the shared shared solution solution and and interfac interfacialial properties properties described described abov abovee [25]. [Our25]. recent Our recent work workexplains explains the high the diversity high diversity of Class of I Classsequences I sequences [25]. To [better25]. Tocontextualiz better contextualizee the primary the structures primary structuresstudied herein, studied we herein, provide we provide a visual a visualrepresentation representation of this of thisanalysis analysis (i.e., (i.e., a asequence sequence alignment principalprincipal component analysis) as it it was was conducted conducted in in [25] [25] in in Figure Figure 22.. Briefly, Briefly, we we constructed constructed a adatabase database of ofconfirmed confirmed and and predicted predicted hydrophobins hydrophobins from from the thegenomes genomes of over of over 200 fungal 200 fungal species. species. The Theprincipal principal component component analysis analysis (PCA) (PCA) of ofthe the protein protein sequence sequence alignment alignment matrix matrix of of the databasedatabase combinedcombined withwith PFamPFam domain domain assignations assignations revealed revealed two two high-identity high-identity Class Class I subdivisions I subdivisions (verified (verified by proteinby protein sequence sequence alignments) alignments) [25 []:25]: Class Class IA, IA, containing containing sequences sequences of of Ascomycota Ascomycota origin, origin, andand ClassClass IB,IB, containingcontaining thosethose ofof BasidiomycotaBasidiomycota origin.origin. The Class I sequences that remain are a mixturemixture ofof AscomycotaAscomycota and BasidiomycotaBasidiomycota sequences that share far lower similarity and lie at thethe interfaceinterface ofof thesethese twotwo groups.groups. We referrefer toto thesethese asas IM sequence sequencess (of mixed phylum), and their subdivisions within ClassClass II areare yetyet toto bebe understood.understood. These groups can be visualized in the plot of thethe firstfirst andand