EDITORIAL published: 30 November 2020 doi: 10.3389/fmicb.2020.620239 Editorial: Protein Aggregation and Solubility in Microorganisms (Archaea, Bacteria and Unicellular Eukaryotes): Implications and Applications Georgios Skretas 1* and Salvador Ventura 2* 1 Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece, 2 Departament de Bioquimica i Biologia Molecular, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain Keywords: protein folding, protein solubility, protein aggregation, recombinant protein production, microorganisms, pathogenic amyloids, functional amyloids Edited by: Editorial on the Research Topic Alberto A. Iglesias, CONICET Coastline Agrobiotechnology Institute Protein Aggregation and Solubility in Microorganisms (Archaea, Bacteria and Unicellular (IAL), Argentina Eukaryotes): Implications and Applications Reviewed by: Héctor M. Alvarez, Proteins constitute the functional molecules of life. Typically, only folded and soluble proteins Consejo Nacional de Investigaciones are functional, whereas the failure of a protein to adopt its intended three-dimensional structure Científicas y Técnicas (protein misfolding) usually results in the formation of biologically inactive, insoluble protein (CONICET), Argentina aggregates. Thus, protein solubility is critical for the maintenance of healthy cellular physiology Leszek A. Kleczkowski, and a major concern for both basic biomedical research and the biotechnology industry. At the Umeå University, Sweden same time, protein misfolding and aggregation have emerged as common pathological features of *Correspondence: more than 50 human diseases, which can result in neurodegeneration, metabolic dysfunction, heart Georgios Skretas and kidney failure, blindness, and other maladies (Chiti and Dobson, 2017). gskretas@eie.gr Salvador Ventura When isolated recombinant proteins are needed for biochemical/structural studies, and as salvador.ventura@uab.cat industrial bioproducts, microbial cell hosts are typically the first choice as protein production factories (Makino et al., 2011a). Their industrial importance is well-reflected by the fact that Specialty section: approximately half of all approved recombinant biopharmaceuticals are produced in unicellular This article was submitted to hosts, such as Escherichia coli and Saccharomyces cerevisiae (Ferrer-Miralles et al., 2009). In this Microbial Physiology and Metabolism, Research Topic, Kang and Seong describe how microbial hosts can be used to produce antibody a section of the journal fragments, a special and important class of biotherapeutic molecules (Kang and Seong). Indeed, Frontiers in Microbiology six of the currently marketed antibody fragment pharmaceuticals, either in the single-chain Received: 22 October 2020 variable fragment (scFv) or the fragment antigen binding (Fab) format, are produced in E. coli. Accepted: 12 November 2020 Unfortunately, a significant proportion of antibody fragment sequences suffer from low solubility, Published: 30 November 2020 low stability, or high-aggregation propensity. The authors present a thorough review of currently Citation: utilized strategies for addressing these issues in microbial expression hosts. These include protein Skretas G and Ventura S (2020) engineering approaches, such as rational mutagenesis, computational design, directed evolution, Editorial: Protein Aggregation and and use of solubility-enhancing fusion partners, as well as cell host engineering techniques, Solubility in Microorganisms (Archaea, Bacteria and Unicellular Eukaryotes): primarily through molecular-chaperone co-expression. Since microbial hosts can also be used to Implications and Applications. produce and to engineer full-length monoclonal antibodies (Mazor et al., 2007; Makino et al., Front. Microbiol. 11:620239. 2011b), it is conceivable that the list of microbially produced marketed biopharmaceuticals could doi: 10.3389/fmicb.2020.620239 soon be expanded to include these molecules as well. Frontiers in Microbiology | www.frontiersin.org 1 November 2020 | Volume 11 | Article 620239 Skretas and Ventura Editorial: Protein Aggregation in Microorganisms Upon production in microbial factories, many recombinant a role of premature IAPP forms in the etiology of type polypeptides undergo irregular or incomplete folding processes 2 diabetes. that usually result in their accumulation as insoluble aggregates, Protein aggregation is a hallmark of many diseases; however, also known as inclusion bodies (IBs). Until very recently, this it is becoming increasingly evident that living systems -and phenomenon was perceived merely as an unwanted side-effect of especially microorganisms- can exploit the phenomenon of recombinant production, and IBs as a kind of useless “dust balls.” protein aggregation and amyloid formation for their own The quite surprising observation, however, that certain microbial benefit. This type of amyloid aggregates is generally referred aggregates can contain a fraction of molecules with native- to as “functional amyloids” (Otzen and Riek, 2019). One such like structure, which can retain biological activity, provided the example of functional amyloid is the CsgA protein of E. coli, motivation to exploit this initially unwanted phenomenon for whose regulated aggregation leads to amyloid formation and understanding the relationship between conformational protein enables this enterobacterium to form highly rigid and resistant quality and solubility, as well as for the development of useful new extracellular biofilms. These biofilms endow E. coli colonies applications (Garcia-Fruitos et al., 2005; de Marco et al., 2019). with a very effective defense mechanism against external attacks, Based on these, Panda and co-workers set out to investigate the such as antibiotic treatment. Thus, the aggregation of CsgA effect of varying the expression temperature on the formation, and other similar microbial proteins are major targets for the morphology and the biological activity of bacterial IBs Singh antibiotic discovery. In an attempt to provide an easy-to-work- et al.. By using the E. coli asparaginase II, a tetrameric enzyme, with starting point for the screening of chemical or biological which can retain enzymic activity in inclusion body form, agents that inhibit CsgA aggregation and biofilm formation, they have made a quite unanticipated observation: the specific Matthew Chapman and his colleagues engineered a double catalytic activity of asparaginase inclusion bodies increases as the cysteine variant of the protein, termed CsgACC , where the Cys production temperatures increases. This observation contradicts residues have been introduced into a region of the protein that is previous studies with other model proteins, where the opposite critical for amyloid formation (Balistreri et al.). Under oxidizing effects were noted, and suggests that the relationship between conditions, CsgACC was shown to adopt a conformation that solubility, aggregation, and conformational quality may be contains a non-native disulfide, which prevents aggregation of protein specific. the protein. Upon addition of a reducing agent, however, this An additional exciting property of proteins embedded covalent bond is broken and CsgA proceeds to a native-like in microbial deposits is that they resemble the amyloid aggregation pathway, resulting in amyloid aggregates, which are fibrils formed in several human neurodegenerative and other virtually indistinguishable from those of the wild-type protein. protein aggregation diseases (Wang et al., 2008). This feature CsgA sequence is highly aggregation-prone and not easy to makes microbial cells appropriate model systems for studying handle, as it is constantly changing its polymerization state. In the mechanisms of pathogenic protein aggregation in an contrast, in CsgACC, the aggregation reaction can be controlled environment that is very simple and, at the same time, more chemically, offering a convenient starting point for screening biologically relevant than a test tube (Dasari et al., 2011; Villar- expeditions looking for CsgA aggregation inhibitors. Pique and Ventura, 2012). Furthermore, it can allow the rapid Due to their central role in biology, protein folding, and screening of therapeutic drug candidates (Villar-Pique et al., solubility are controlled genetically, transcriptionally, and at 2012). In some instances, microbes can be even engineered to the protein sequence and structural level. Besides, a well- autonomously perform both the production of the molecular conserved cellular machinery assists the folding of polypeptides libraries under investigation and their screening for aggregation- and preserves proteostasis. Molecular chaperones, such as the inhibitory activity (Matis et al., 2017; Delivoria et al., 2019). Yeast ones belonging to the heat shock protein 70 (Hsp70) and cells, in particular, are also excellent systems for conveniently Hsp90 families play a central role in the maintenance of studying the link between protein aggregation and cytotoxicity proper protein folding, solubility, and, if needed, controlled (Kostelidou et al., 2018). In this direction, Regina Menezes’ lab clearance. To assist these major cellular folding factors and used Saccharomyces cerevisiae as a model organism to study the for specialized missions, cells also use the so-called small heat properties of different forms of the islet amyloid polypeptide shock proteins (Hsps). One example of them is Hsp33, a redox (IAPP), a protein whose aggregation can be toxic for pancreatic state-regulated molecular chaperone that promotes bacterial β-cells and contribute to the pathology of type 2 diabetes and algae survival under heat and oxidative stress conditions. Raimundo et al.. IAPP, also known as amylin, is a polypeptide The laboratory of Dana Reichmann has applied bioinformatic whose mature form is produced as a result of the sequential analyses to identify new Hsp33 candidates and have found that, proteolytic processing of the initial pre-pro-IAPP sequence. apart from bacteria and algae, similar sequences can be found Raimundo et al. used yeast cells to investigate the aggregation and in eukaryotic organisms, mainly in unicellular parasites and subsequent cytotoxicity of premature forms of IAPP compared organisms residing in extreme environments (Aramin et al.). to its mature form. These investigators have found that the initial Based on this observation, they investigated whether Hsp33 could precursor polypeptide pre-pro-IAPP is particularly toxic and that be an essential factor for the survival of these eukaryotic microbes this toxicity is associated with the intracellular accumulation under harsh living conditions. They chose to study the Hsp33 of insoluble oligomeric species. These observations point to protein from Trypanosoma brucei, a eukaryotic pathogen causing Frontiers in Microbiology | www.frontiersin.org 2 November 2020 | Volume 11 | Article 620239 Skretas and Ventura Editorial: Protein Aggregation in Microorganisms African sleeping sickness. They called this protein TrypOx and science as well as new tools for drug discovery/development and investigated whether this parasite uses this factor to prevent its other important biotechnological applications. proteins from misfolding and as a defensive strategy against the oxidative stress attack exerted by the immune system of the infected host, in this case human cells. Aramin et al. have found AUTHOR CONTRIBUTIONS that TrypOx is indeed a redox sensitive protein that, through GS and SV wrote the paper. All authors have read and approved interaction with the main Hsp70/Hsp40 chaperone complex, the final version of the manuscript. assists protein folding and contributes to T. brucei survival under oxidative stress. Thus, TrypOx and, by extension, the Hsp33 proteins from other eukaryotic pathogens may be relevant targets FUNDING for drug discovery. Overall, the study of protein folding, solubility, and GS was supported by the European Research Council (ERC) aggregation in all types of microorganisms (archaea, bacteria, under the European Union’s Horizon 2020 research and and unicellular eukaryotes) is an exciting and increasingly innovation program (Project ProMiDis; grant agreement vibrant field that can provide both important insights in basic no 819934). REFERENCES Makino, T., Skretas, G., Kang, T., H., and Georgiou, G. (2011b). Comprehensive engineering of Escherichia coli for enhanced expression of IgG antibodies. Chiti, F., and Dobson, C. M. (2017). Protein misfolding, amyloid formation, Metab. Eng. 13, 241–251. doi: 10.1016/j.ymben.2010.11.002 and human disease: a summary of progress over the last decade. Annu. Rev. Matis, I., Delivoria, D., C., Mavroidi, B., Papaevgeniou, N., Panoutsou, S., Biochem. 86, 27–68. doi: 10.1146/annurev-biochem-061516-045115 et al. (2017). An integrated bacterial system for the discovery of chemical Dasari, M., Espargaro, A., Sabate, R., Lopez del Amo, J. M., Fink, U., Grelle, G., rescuers of disease-associated protein misfolding. Nat. Biomed. Eng. 1, 838–852. et al. (2011). Bacterial inclusion bodies of Alzheimer’s disease beta-amyloid doi: 10.1038/s41551-017-0144-3 peptides can be employed to study native-like aggregation intermediate states. Mazor, Y., Van Blarcom, T., Mabry, R., Iverson, B., L., and Georgiou, G. (2007). ChemBiochem 12, 407–423. doi: 10.1002/cbic.201000602 Isolation of engineered, full-length antibodies from libraries expressed in de Marco, A., Ferrer-Miralles, N., Garcia-Fruitos, E., Mitraki, A., Peternel, S., Escherichia coli. Nat. Biotechnol. 25, 563–565. doi: 10.1038/nbt1296 Rinas, U., et al. (2019). Bacterial inclusion bodies are industrially exploitable Otzen, D., and Riek, R. (2019). Functional amyloids. Cold Spring Harb. Perspect amyloids. FEMS Microbiol. Rev. 43, 53–72. doi: 10.1093/femsre/fuy038 Biol. 11, 1–28. doi: 10.1101/cshperspect.a033860 Delivoria, D. C., Chia, S., Habchi, J., Perni, M., Matis, I., Papaevgeniou, N., Villar-Pique, A., Espargaro, A., Sabate, R., SdeGroot, N., and Ventura, S. (2012). et al. (2019). Bacterial production and direct functional screening of expanded Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors. molecular libraries for discovering inhibitors of protein aggregation. Sci. Adv. Microb. Cell Fact 11:55. doi: 10.1186/1475-2859-11-55 5:eaax5108. doi: 10.1126/sciadv.aax5108 Villar-Pique, A., and Ventura, S. (2012). Modeling amyloids in bacteria. Microb. Ferrer-Miralles, N., Domingo-Espin, J., Corchero, J. L., Vazquez, E., and Villaverde, Cell. Fact 11:166. doi: 10.1186/1475-2859-11-166 A. (2009). Microbial factories for recombinant pharmaceuticals. Microb. Cell Wang, L., Maji, S., K., Sawaya, M., R., Eisenberg, D., et al. (2008). Fact 8:17. doi: 10.1186/1475-2859-8-17 Bacterial inclusion bodies contain amyloid-like structure. PLoS Biol. 6:e195. Garcia-Fruitos, E., Gonzalez-Montalban, N., Morell, M., Vera, A., Ferraz, R., doi: 10.1371/journal.pbio.0060195 M., et al. (2005). Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb Cell Fact 4:27. Conflict of Interest: The authors declare that the research was conducted in the doi: 10.1186/1475-2859-4-27 absence of any commercial or financial relationships that could be construed as a Kostelidou, K., Matis, I., and Skretas, G. (2018). Microbial genetic screens for potential conflict of interest. monitoring protein misfolding associated with neurodegeneration: tools for identifying disease-relevant genes and for screening synthetic and natural Copyright © 2020 Skretas and Ventura. This is an open-access article distributed compound libraries for the discovery of potential therapeutics. Curr. Pharm. under the terms of the Creative Commons Attribution License (CC BY). The use, Des. 24, 2055–2075. doi: 10.2174/1381612824666180515143752 distribution or reproduction in other forums is permitted, provided the original Makino, T., Skretas, G., and Georgiou, G. (2011a). Strain engineering for improved author(s) and the copyright owner(s) are credited and that the original publication expression of recombinant proteins in bacteria. Microb. Cell Fact 10:32. in this journal is cited, in accordance with accepted academic practice. No use, doi: 10.1186/1475-2859-10-32 distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 3 November 2020 | Volume 11 | Article 620239