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

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.

Characteristics and Functions of Hydrophobins and Their Use in Manifold Industries

“…It should be noted that with the decrease in sequencing costs, each year more fungal genomes are discovered, which is why the current division may become out of date. The search of databases confirms that class II hydrophobins are present only in Ascomycota as a homogeneous group in the phylogenetic tree [23,59] (Fig. 2).…”

“…The research conducted on the HGFI hydrophobin from Grifola frondosa and hydrophobins produced by Pleurotus ostreatus using an atomic force microscope (AFM) has shown that the length of tubular fibres is from 50 to 105 nm, and the width ranges from 19 to 24 nm. The rodlet layers take the form of a double layer [23,41,49,88,89]. In 2013, Stanimirova et al [68] conducted a series of experiments aimed at demonstrating changes in surface pressure during the formation of the hydrophobic film.…”

“…Badania prowadzone nad hydrofobiną HGFI pochodzącą z żagwicy listkowatej (Grifola frondosa) oraz hydrofobinami wytwarzanymi przez boczniaka ostrygowatego (Pleurotus ostreatus) z wykorzystaniem mikroskopii sił atomowych (AFM, Atomic Force Microscope) wykazały, że długość włókien rurkowatych wynosi od 50 do 105 nm, a szerokość waha się w zakresie od 19 do 24 nm. Powierzchnie rurkowate przyjmują formę podwójnej warstwy [23,41,49,88,89]. W 2013 roku Stanimirova i wsp.…”

“…Klasę Ia cechuje większe zróżnicowanie długości sekwencji genów kodujących te białka. Hydrofobowość większości białek klasy Ib jest średnio wyższa o 60% w porównaniu z hydrofobinami klasy Ia i klasy II[23, 59].…”

The Use of α-MF Signal Peptide Without Spacer for Producing Insulin Aspart Precursor in Pichia pastoris KM71

“…The phenotype of colony used for insulin Aspart precursor production is Mut S which has slow ability to utilize methanol as sole carbon source. Mut S phenotype enables methanol accumulation for promoter activation in shake flask culture since the methanol is supplied once daily (Gandier and Master, 2018). The provided methanol in the medium will not be rapidly utilized to accelerate its growth but will be used instead for promoter induction.…”