Genetic Engineering of Filamentous Fungi for Efficient Protein Expression and Secretion

Wang, Qin; Zhong, Chao; Xiao, Han

doi:10.3389/fbioe.2020.00293

Cited by 70 publications

(69 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Data obtained here showed that expression of chimeric protein in lower temperature (25-26 o C) and adding 40% more YNB culture medium into the main culture medium (BMY) was more e cient approach for protein expression than prevailing approaches such as stated in Invitrogen protocol (Safder et al .2018). It might be due to the positive effect of low temperature and nutritional supplementation on protein expression and could be considered for large-scale heterogeneous protein performance (Silverman et al2017;Mori et al 2017;Wang et al .2020) One of the major challenges in producing bio-adhesives from mussel foot proteinosis is adhesive proteins demonstrating resistance at a particular pH. In the present study, AFM microscopic images also show greater cohesion of proteins in acidic pH.…”

Section: Discussionsupporting

confidence: 54%

A Novel Recombinant Chimeric Proteinous Bio-adhesive Consisting of Mussel Foot Protein 3, 5 and Curli Proteins GvpA and CsgA Expressed in Pichia Pastoris

Bolghari

Habibollahi

Anvari

et al. 2021

Preprint

View full text Add to dashboard Cite

Despite various efforts to produce strong recombinant bio-adhesive proteins for medical purposes, efficient production of a safe and feasible bio-glue is not yet a commercial reality due to the weak properties or low expression level. Here, a feasible expression system has been developed to produce strong recombinant fusion protein using Mfp3 and Mfp5 along with two curli proteins, GvpA and CsgA expressed under the control of alcohol oxidase (AOX1) promoter for high-level production in P. pastoris using pPICZα vector. Purified chimeric proteins were first evaluated using western blotting and its remaining dopa were measured in the modified proteins by NBT assay. We further elucidated mechanistic properties of obtained adhesive protein assembly in various pH based on its different subunits using atomic force microscopy (AFM) when adsorbed onto the mica surface. We found that combinational structural features of subunits and post-translational changes during expression in yeast host have led to potent adherence due to higher dopa residues majorly in acidic condition and tetrad complex that is higher than that of earlier reports in prokaryotic systems. We believe that our obtained chimeric protein resulted through fusion of GvpA and CsgA proteins with dopa-containing Mfp proteins expressed in the methylotrophic yeast, P. pastoris, not only presents a candidate for future biomedical applications, but also provides novel biological clues using for high-performance bioinspired biomaterials designation.

show abstract

Section: Discussionsupporting

confidence: 54%

A Novel Recombinant Chimeric Proteinous Bio-adhesive Consisting of Mussel Foot Protein 3, 5 and Curli Proteins GvpA and CsgA Expressed in Pichia Pastoris

Bolghari

Habibollahi

Anvari

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…In general, secretory systems are advantageous because the protein product is exported into the medium allowing for economic and straightforward downstream processing workflows [ 8 ]. Due to their extraordinary secretion capacities and inexpensive cultivation, fungal expression hosts are promising candidates for novel platforms and already the preferred hosts for the production of proteases and other hydrolytic enzymes [ 9 , 10 ]. However, the synthesis of heterologous proteins still imposes major challenges in fungal expression hosts [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

Hussnaetter

Philipp

Müntjes

et al. 2021

JoF

View full text Add to dashboard Cite

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

show abstract

“…Filamentous fungi are the only microorganisms that can fully degrade renewable lignocellulosic biomass and sustainably transform it into food, feed, chemicals, fuels, commodities, textiles, composite materials, antibiotics, and other drugs (Meyer et al, 2020). Their ability to perform complex posttranslational modifications (Wang et al, 2020), their greatly expanded natural protein secretion apparatus (Ward, 2012), and the enormous potential of the secondary metabolome to produce bioactive molecules (Brakhage, 2013; Keller, 2019; Nielsen et al, 2017) make filamentous fungi a favorable host for many applications (Wösten, 2019).…”

Section: Introductionmentioning

confidence: 99%

Universal law for diffusive mass transport through mycelial networks

Schmideder

Müller

Barthel

et al. 2020

Biotech & Bioengineering

View full text Add to dashboard Cite

Filamentous fungal cell factories play a pivotal role in biotechnology and circular economy. Hyphal growth and macroscopic morphology are critical for product titers; however, these are difficult to control and predict. Usually pellets, which are dense networks of branched hyphae, are formed during industrial cultivations. They are nutrient‐ and oxygen‐depleted in their core due to limited diffusive mass transport, which compromises productivity of bioprocesses. Here, we demonstrate that a generalized law for diffusive mass transport exists for filamentous fungal pellets. Diffusion computations were conducted based on three‐dimensional X‐ray microtomography measurements of 66 pellets originating from four industrially exploited filamentous fungi and based on 3125 Monte Carlo simulated pellets. Our data show that the diffusion hindrance factor follows a scaling law with respect to the solid hyphal fraction. This law can be harnessed to predict diffusion of nutrients, oxygen, and secreted metabolites in any filamentous pellets and will thus advance the rational design of pellet morphologies on genetic and process levels.

show abstract

Genetic Engineering of Filamentous Fungi for Efficient Protein Expression and Secretion

Cited by 70 publications

References 73 publications

A Novel Recombinant Chimeric Proteinous Bio-adhesive Consisting of Mussel Foot Protein 3, 5 and Curli Proteins GvpA and CsgA Expressed in Pichia Pastoris

A Novel Recombinant Chimeric Proteinous Bio-adhesive Consisting of Mussel Foot Protein 3, 5 and Curli Proteins GvpA and CsgA Expressed in Pichia Pastoris

Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

Universal law for diffusive mass transport through mycelial networks

Contact Info

Product

Resources

About