Abstract
Hydrophobins are small, surface-active protein biosurfactants secreted by filamentous fungi with various applications in industries such as pharmaceuticals, sanitation, and biomaterials. Their amphiphilic structure enables micelle formation for drug delivery, and their thermal stability allows low-temperature detergent formulation for use in cleaning agents. Additionally, hydro-phobins are known to stabilize enzymatic processes for improved catalytic efficiency, creating an opportunity to economically produce value-added material from biomass. Trichoderma reesei, a well-characterized fungal system used industrially for bioethanol production, co-secretes a hy-drophobin, HFBI, alongside cellulose-degrading enzymes during biomass turnover. Prior studies suggest hydrophobins modify lignocellulosic surface features upon binding to enhance biomass decomposition, presenting HFBI as a green alternative to industrially widespread petrochemical surfactants utilized in production of crystalline nanocellulose (CNC). CNC is a powerful biomaterial for direct substitution in construction, automotive, and photonic materials, but production is plagued by high environmental costs and low yields, the latter of which has been mitigated by exacerbating the former through introduction of petrochemical surfactants for improved cellulose processing. This work presents hydrophobins as a green, tailored-by-nature replacement for petrochemical sur-factants to improve enzymatic performance while reducing environmental impacts of CNC pro-duction. In this dissertation I use Pichia pastoris to recombinantly overproduce hydrophobin HFBI from Trichoderma reesei. I iteratively optimize both the induction and purification of HFBI, ultimately producing yields of 86.6 mg/L HFBI and elution concentrations of 48 μM HFBI with a single unit operation multimodal cation exchange purification. I express an evolutionary library of HFBI variants on the yeast cell surface based on sequence conservation to screen for improved binding behavior to fluorescently labeled cellulose via fluorescence-activated cell sorting (FACS). Despite the inadvertent use of zeocin selection, seven highly fluorescent mutants are successfully isolated from one million sorted. Through secondary screening for correct protein size, one of these mutants is selected for sequencing. Upon transformation into P. pastoris for secretion, this mutant is shown to outperform wild type HFBI in cellulose binding assays. Lastly, I explore some applications beyond CNC production, replacing petrochemical surfactants in food packaging and concrete with HFBI. The results of this dissertation lead to improved understanding of scalable HFBI production and purification, directed evolution of HFBI for improved cellulose binding, and HFBI applications replacing petrochemical surfactants.
